Single variable domain immunoglobulins that bind TGF-beta receptor II

ABSTRACT

The disclosure provides an anti-TGFbetaRII immunoglobulin single variable domain. Suitably, an anti-TGFbetaRII immunoglobulin single variable domain in accordance with the disclosure is one having an amino acid sequence as set forth in any one of SEQ ID NO:1-28 having up to 5 amino acid substitutions, deletions or additions. The disclosure further provides a polypeptide and pharmaceutical composition for treating a disease associated with TGFbeta signalling and suitably a disease selected from the group of: tissue fibrosis, such as pulmonary fibrosis, including idiopathic pulmonary fibrosis; liver fibrosis, including cirrhosis and chronic hepatitis; rheumatoid arthritis; ocular disorders; fibrosis of the skin, including keloid of skin; Dupuytren&#39;s Contracture; kidney fibrosis such as nephritis and nephrosclerosis; wound healing; scarring reduction; and a vascular condition, such as restenosis.

This application is a 371 of International Application No.PCT/EP2012/050061, filed 4 Jan. 2012, which claims the benefit of U.S.Provisional Application No. 61/430,235, filed 4 Jan. 2011, both of whichare herein incorporated by reference in their entireties.

BACKGROUND

Transforming Growth Factor-β (TFGbeta; TGFβ (TGF-β) is a signalingmolecule that mediates signal transduction into cells through binding toa TGFbeta receptor (TGFbetaR; TGFβR (TGF-βR)). TGFbeta signalingactivity regulates cell differentiation and growth, the nature of itseffect, i.e. as cell growth-promoter, growth-suppressor or inducer ofother cell functions, being dependent on cell type (see Roberts, et al.,The transforming growth factor-betas, Peptide Growth Factors and TheirReceptors, Part I, ed. by Sporn, M. B. & Roberts, A. B.,Springer-Verlag, Berlin, 1990, p. 419-472).

TGFbeta is produced by a wide variety of cell types, and its cognatereceptors are expressed in a wide variety of organs and cells (see Shiand Massague, Cell, Volume 113, Issue 6, 13 Jun. 2003, Pages 685-700;Biol. Signals., Vol. 5, p. 232, 1996 and Pulmonary Fibrosis, Vol. 80 ofLung Biology in Health and Disease Series, ed. by Phan, et al., p. 627,Dekker, New York, 1995). TGFbeta receptors have been identified to fallinto three types: TGFbetaRI (TGFβRI) (TGFbeta type I receptor (Franzenet al., Cell, Vol. 75, No. 4, p. 681, 1993; GenBank Accession No:L11695)); TGFbetaRII (TGFβRII) (TGFbeta type II receptor (Herbert etal., Cell, Vol. 68, No. 4, p. 775, 1992; GenBank Accession No: M85079))and TGFbetaRIII (TGFbeta type III receptor (Lopez-Casillas, Cell, Vol.67, No. 4, p. 785, 1991; GenBank Accession No: L07594)). TGFbetaRI andTGFbetaRII have been shown to be essential for the signal transductionof TGF-beta (Laiho et al., J. Biol. Chem., Vol. 265, p. 18518, 1990 andLaiho et al., J. Biol. Chem., Vol. 266, p. 9108, 1991), whileTGFbetaRIII is not thought to be essential.

TGFbeta signaling is mediated through its binding to both TGFbetaRI andRII. When the ligand binds to the extracellular ligand binding domain,the two receptors are brought together, allowing RII to phosphorylate RIand begin the signaling cascade through the phosphorylation of Smadproteins (see Shi and Massague as referred to above).

Three isoforms of TGFbeta have been identified in mammals: TGFbeta1,TGFbeta2, and TGFbeta3. Each isoform is multifunctional and acts inself-regulatory feedback mechanisms to control bioavailability fordevelopmental processes and to maintain tissue homeostasis (as reviewedin ten Dijke and Arthur, Nature Reviews, Molecular Cell Biology, Vol. 8,November 2007, p. 857-869). Levels of TFGbeta are controlled byregulation through TGFbeta expression as well as through binding toproteoglycan, i.e., the extracellular matrix (ECM).

Dysregulated TGFbeta signaling, such as excess TGFbeta signaling andhigh levels of bioavailable TGFbeta, is implicated in a number ofpathologies, including fibroses of various tissues, such as pulmonaryfibrosis and cirrhosis, chronic hepatitis, rheumatoid arthritis, oculardisorders, vascular restenosis, keloid of skin, and the onset ofnephrosclerosis.

Accordingly, there is a need to provide compounds that block or disruptTGFbeta signaling in a specific manner, such as through binding to theTGFbeta receptor II. Such compounds can be used in therapeutics.

SUMMARY

The disclosure relates to an anti-TGFbetaRII immunoglobulin singlevariable domain. Suitably, an anti-TGFbetaRII immunoglobulin singlevariable domain in accordance with the disclosure is one which binds toTGFbetaRII with an equilibrium dissociation constant (KD) in the rangeof 10 pM to 50 nM, optionally 10 pM to 10 nM, optionally 100 pM to 10nM. In one embodiment, the anti-TGFbetaRII immunoglobulin singlevariable domain is one which binds TGFbetaRII with high affinity (highpotency) and has an equilibrium dissociation constant of 10 pM to 500pM. In one embodiment, the anti-TGFbetaRII immunoglobulin singlevariable domain is one which binds TGFbetaRII with an affinity (KD) ofapproximately 100 pM. In one embodiment, the anti-TGFbetaRIIimmunoglobulin single variable domain is one which binds TGFbetaRII withan affinity (KD) of less than 100 pM. In another embodiment, theanti-TGFbetaRII immunoglobulin single variable domain is one which bindsTGFbetaRII with moderate affinity (low potency) and has an equilibriumdissociation constant of 500 pM to 50 nM, preferably 500 pM to 10 nM. Inanother aspect, the disclosure provides an isolated polypeptidecomprising an anti-TGFbetaRII immunoglobulin single variable domain.Suitably, the isolated polypeptide binds to human TGFbetaRII. In anotherembodiment, the isolated polypeptide also binds to TGFbetaRII derivedfrom a different species such as mouse, dog or monkeys, such ascynomolgus monkeys (cyno). Suitably, the isolated polypeptide binds toboth mouse and human TGFbetaRII. Such cross reactivity betweenTGFbetaRII from humans and other species allows the same antibodyconstruct to be used in an animal disease model, as well as in humans.

In an aspect of the disclosure there is provided an anti-TGFbetaRIIimmunoglobulin single variable domain having an amino acid sequence asset forth in any one of SEQ ID NO:1-38, 204, 206, 208, 214, 234, 236,238, 240, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and287, and having up to 5 amino acid alterations, wherein each amino acidalteration is an amino acid substitution, deletion or addition i.e. upto 5 amino acid substitutions, deletions or additions, in anycombination. In a particular embodiment the amino acid substitutions areconservative substitutions.

In an embodiment, the anti-TGFbetaRII immunoglobulin single variabledomain has the amino acid sequence as set forth in SEQ ID NO: 234 or 279and having up to 5 amino acid alterations, wherein each amino acidalteration is a an amino acid substitution, deletion or addition. In aparticular embodiment, the amino acid alteration(s) are not within CDR3,more specifically not within CDR3 and CDR1, or CDR3 and CDR2, morespecifically not within any of the CDRs. In an embodiment, theanti-TGFbetaRII immunoglobulin single variable domain consists of anyone of the following sequences: SEQ ID NO:1-38, 204, 206, 208, 214, 234,236, 238, 240, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283,285 and 287. In an embodiment the anti-TGFbetaRII immunoglobulin singlevariable domain consists of an amino acid sequence of SEQ ID NO: 234 or236.

It is not intended to cover any specific anti-TGFbetaRII immunoglobulinsingle variable domain sequence disclosed in WO 2011012609. For theavoidance of doubt each and every sequence disclosed in WO 2011012609may be disclaimed from the present invention. In particular DOM23h-271(SEQ ID NO:4) and DOM-23h-439 (SEQ ID NO:10) as disclosed in WO2011012609 may be disclaimed. An anti-TGFbetaRII immunoglobulin singlevariable domain consisting of the amino acid sequence as set forth inSEQ ID NO: 199 or 201 herein may be disclaimed.

An anti-TGFbetaRII immunoglobulin single variable domain according tothe invention may comprise one or more (e.g. 1, 2, 3, 4, or 5)C-terminal alanine residues. Alternatively, an anti-TGFbetaRIIimmunoglobulin single variable domain may comprise a C-terminal peptideof up to 5 amino acids in length. In an embodiment, the C-terminalpeptide comprises 1, 2, 3, 4, or 5 amino acids.

A person skilled in the art is able to deduce from a given singlevariable domain sequence, e.g. one having a sequence as set out in anyone of SEQ ID NO:1-38, 204, 206, 208, 214, 234, 236, 238, 240, 263, 265,267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287 which CDRsequences are contained within them using the various methods outlinedherein e.g. CDR sequences as defined by reference to Kabat (1987),Chothia (1989), AbM or contact methods, or a combination of thesemethods. Suitably, CDR sequences are determined using the method ofKabat described herein. In one embodiment, the CDR sequences of eachsequence are those set out in tables 1, 2, 9, and 13.

In an aspect of the invention an anti-TGFbetaRII immunoglobulin singlevariable domain of the disclosure has 90% or greater than 90% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NO:1-28.

In an aspect of the invention an anti-TGFbetaRII immunoglobulin singlevariable domain of the disclosure has an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-28 with 25 or fewer amino acidchanges. In a particular embodiment an anti-TGFbetaRII immunoglobulinsingle variable domain of the disclosure has an amino acid sequenceselected from the group consisting of SEQ ID NO:1-28 with 20 or fewer,15 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 orfewer, or 5 or fewer amino acid changes.

In an aspect of the disclosure there is provided an isolated polypeptidecomprising an anti-TGFbetaRII immunoglobulin single variable domain ofthe disclosure, in particular an anti-TGFbetaRII immunoglobulin singlevariable domain identical to an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-38, 204, 206, 208, 214, 234, 236, 238,240, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287wherein said isolated polypeptide binds to TGFbetaRII.

In an aspect of the disclosure there is provided an isolated polypeptideencoded by a nucleotide sequence that is at least 80% identical to atleast one nucleic acid sequence selected from the group of: SEQ IDNOS:39-66, wherein said isolated polypeptide binds to TGFbetaRII.

An anti-TGFbetaRII immunoglobulin single variable domain or apolypeptide in accordance with any aspect of the disclosure may compriseany of the following amino acids: R at position 39, I at position 48, Dat position 53, N at position 61, R at position 61, K at position 61, Rat position 64, F at position 64, D at position 64, E at position 64, Yat position 64, H at position 102, or S at position 103 of theimmunoglobulin single variable domain, said position being according tothe kabat numbering convention. In one embodiment, the immunoglobulinsingle variable domain or polypeptide comprises a combination of theseamino acids. In another embodiment, the immunoglobulin single variabledomain or polypeptide comprises amino acid N at 61 and R at 64. Inanother embodiment, the immunoglobulin single variable domain orpolypeptide comprises amino acid R or K at position 61. In anembodiment, the anti-TGFbetaRII immunoglobulin single variable domaincomprises an I at position 48 in addition to any one of theaforementioned residues at position 61 and/or 64. In these embodiments,the amino acid numbering is that of the immunoglobulin single variabledomain, as exemplified, for example, by those sequences given in SEQ IDNOs:1-38, 204, 206, 208, 214, 234, 236, 238, 240, 263, 265, 267, 269,271, 273, 275, 277, 279, 281, 283, 285 and 287.

An anti-TGFbetaRII immunoglobulin single variable domain or apolypeptide in accordance with any aspect of the disclosure may compriseone of the following amino acid combinations selected from the group: Nat position 61 and R at position 64; R at position 61 and E at position64; R at position 61 and M at position 64; R at position 61 and F atposition 64; R at position 61 and Y at position 64; and R at position 61and D at position 64 of the immunoglobulin single variable domain. In anembodiment, the anti-TGFbetaRII immunoglobulin single variable domaincomprises an I at position 48 in addition to any one of theaforementioned combination of residues at positions 61 and 64.

In another aspect, there is provided a ligand or binding moiety that hasbinding specificity for TGFbetaRII and inhibits the binding of ananti-TGFbetaRII immunoglobulin single variable domain comprising anamino acid sequence selected from the group of SEQ ID NOs:1-28 toTGFbetaRII.

In a further aspect of the disclosure, there is provided a fusionprotein comprising an immunoglobulin single variable domain, polypeptideor ligand in accordance with any aspect of the disclosure.

In one embodiment, the immunoglobulin single variable domain,polypeptide, ligand or fusion protein in accordance with the disclosureis one which neutralises TGFbeta activity. Suitably, the immunoglobulinsingle variable domain or polypeptide in accordance with the disclosureinhibits binding of TGFbeta to TGFbetaRII. In another embodiment, theimmunoglobulin single variable domain or polypeptide in accordance withthe disclosure inhibits TGFbeta signalling activity through TGFbetaRII.In another embodiment, the immunoglobulin single variable domain orpolypeptide in accordance with the disclosure suppresses TGFbetaactivity, in particular, TGFbeta cell growth activity and/or fibrogenicactivity. Suitably, TGFbetaRII is human TGFbetaRII.

In one embodiment, the immunoglobulin single variable domain,polypeptide, ligand or fusion protein in accordance with the disclosureis devoid of TGFbetaRII agonist activity at 15 micromolar (μM).

In another aspect, there is provided an immunoglobulin single variabledomain, polypeptide, ligand or fusion protein in accordance with anyaspect of the disclosure further comprising a half-life extendingmoiety. Suitably, the half-life extending moiety is a polyethyleneglycol moiety, serum albumin or a fragment thereof, transferrin receptoror a transferrin-binding portion thereof, or an antibody or antibodyfragment comprising a binding site for a polypeptide that enhanceshalf-life in vivo. In one embodiment, the half-life extending moiety isan antibody or antibody fragment comprising a binding site for serumalbumin or neonatal Fc receptor. In another embodiment, the half-lifeextending moiety is a dAb, antibody or antibody fragment.

In another aspect, the disclosure provides an isolated or recombinantnucleic acid encoding a polypeptide comprising an anti-TGFbetaRIIimmunoglobulin single variable domain, polypeptide, ligand or fusionprotein in accordance with any aspect of the disclosure.

In one embodiment, the isolated or recombinant nucleic acid moleculecomprises or consists of a nucleic acid molecule selected from the groupof any of the nucleic acid molecules having the sequences set out in SEQID NOS:39-76, 203, 205, 207, 212, 233, 235, 237, 239, 262, 264, 266,268, 270, 272, 274, 276, 278, 280, 282, 284, 286.

In one aspect, the disclosure provides an isolated or recombinantnucleic acid, wherein the nucleic acid comprises a nucleotide sequencethat is at least 80% identical to the nucleotide sequence of any of thenucleic acid molecules having the sequences set out in SEQ ID NOS:39-66,and wherein the nucleic acid encodes a polypeptide comprising animmunoglobulin single variable domain that specifically binds toTGFbetaRII.

In another aspect, there is provided a vector comprising a nucleic acidin accordance with the disclosure.

In a further aspect, there is provided a host cell comprising a nucleicacid or a vector in accordance with the disclosure. In yet anotheraspect of the disclosure there is provided a method of producing apolypeptide comprising an anti-TGFbetaRII immunoglobulin single variabledomain or a polypeptide or ligand or a fusion protein in accordance withthe disclosure, the method comprising maintaining a host cell inaccordance with the disclosure under conditions suitable for expressionof said nucleic acid or vector, whereby a polypeptide comprising animmunoglobulin single variable domain, polypeptide or ligand or fusionprotein is produced. Optionally, the method further comprises the stepof isolating the polypeptide and optionally producing a variant, e.g., amutated variant, having an improved affinity (Kd); or EC50 for TGFbetaneutralization in a standard assay than the isolated polypeptide.Suitable assays for TGFbeta activity, such as a cell sensor assay, aredescribed herein, for example, in the Examples section.

In one aspect of the disclosure, the anti-TGFbetaRII immunoglobulinsingle variable domain, polypeptide or ligand or fusion protein inaccordance with the disclosure is for use as a medicament. Accordingly,there is provided a composition comprising anti-TGFbetaRIIimmunoglobulin single variable domain, polypeptide or ligand or fusionprotein in accordance with the disclosure for use as a medicament.

In one aspect of the disclosure, there is provided a use of ananti-TGFbetaRII immunoglobulin single variable domain, polypeptide orligand or fusion protein in accordance with the disclosure for themanufacture of a medicament, particularly for use in treating diseaseassociated with TGFbeta signalling.

Suitably, the anti-TGFbetaRII immunoglobulin single variable domain,polypeptide or ligand or fusion protein or composition in accordancewith the disclosure is for treatment of a disease associated withTGFbeta signaling. Suitably, the disease is a tissue fibrosis, such aspulmonary fibrosis including idiopathic pulmonary fibrosis; liverfibrosis, including cirrhosis and chronic hepatitis; rheumatoidarthritis; ocular disorders; or fibrosis of the skin including keloid ofskin; Dupuytren's Contracture; and kidney fibrosis such as nephritis andnephrosclerosis; or a vascular condition such as restenosis. Otherdiseases associated with TGFbeta signaling include vascular diseasessuch as hypertension, pre-eclampsia, hereditary haemorrhagictelangtiectasia type I (HHT1), HHT2, pulmonary arterial hypertension,aortic aneurysms, Marfan syndrome, familial aneurysm disorder,Loeys-Dietz syndrome, arterial tortuosity syndrome (ATS). Other diseasesassociated with TGFbeta signaling include diseases of themusculoskeletal system, such as Duchenne's muscular dystrophy and musclefibrosis. Further diseases associated with TGFbeta signaling includecancer, such as colon, gastric, and pancreatic cancer, as well as gliomaand NSCLC. In addition, the disclosure provides methods for targetingcancer by modulating TGFbeta signaling in tumour angiogenesis. Otherdiseases or conditions include those related to tissue scarring. Otherdiseases include pulmonary diseases such as COPD (Chronic obstructivepulmonary disease). An anti-TGFbetaRII immunoglobulin single variabledomain, polypeptide or ligand or fusion protein or composition inaccordance with the disclosure may be used in wound healing and/or toprevent or improve the formation of scars. In one aspect, the disclosureprovides the anti-TGFbetaRII single variable domain, ligand orantagonist, composition or fusion protein for intradermal delivery. Inone aspect, the disclosure provides the anti-TGFbetaRII single variabledomain, ligand or antagonist or fusion protein for delivery to the skinof a patient. In one aspect, the disclosure provides the use of theanti-TGFbetaRII single variable domain, ligand or antagonist or fusionprotein in the manufacture of a medicament for intradermal delivery. Inone aspect, the disclosure provides the use of the anti-TGFbetaRIIsingle variable domain or antagonist or fusion protein in accordancewith the disclosure in the manufacture of a medicament for delivery tothe skin of a patient.

In one embodiment, the variable domain is substantially monomeric. In aparticular embodiment the variable domain is 65%-98% monomeric insolution as determined by SEC-MALS. In another embodiment the variabledomain is 65%-100%, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%,95%-100% monomeric in solution as determined by SEC-MALS.

In another embodiment, the variable domain, ligand, fusion protein orpolypeptide as disclosed herein, particularly when in a pharmaceuticalcomposition, does not contain any one or combination or all of thefollowing post-translational modifications: deamidation, oxidation orglycosylation. In a particular embodiment the variable domain, ligand,fusion protein or polypeptide according to the disclosure does notdeamidate.

Suitably, the composition is for therapy or prophylaxis of aTGFbeta-mediated condition in a human.

Accordingly, in one embodiment, there is provided an anti-TGFbetaRII dAbfor treating fibrosis of the skin, in particular keloid disease orDupuytren's Contracture. Suitably, the anti-TGFbetaRII dAb is providedas a substantially monomeric dAb for intradermal delivery, preferablylacking any tag (i.e., untagged) such as a myc or another purificationtag.

In one aspect, the composition is a pharmaceutical composition andfurther comprises a pharmaceutically acceptable carrier, excipient ordiluent.

In another aspect, there is provided a method of treating and/orpreventing an TGFbeta-mediated condition in a human patient, the methodcomprising administering a composition comprising an anti-TGFbetaRIIimmunoglobulin single variable domain, polypeptide or ligand inaccordance with the disclosure the to the patient.

In a further aspect, the disclosure provides an intradermal deliverydevice containing a composition in accordance with the disclosure.Suitably, such a device is a microneedle or collection of microneedles.

An a further aspect, there is provided a kit comprising ananti-TGFbetaRII single variable domain or polypeptide as disclosedherein and a device, such as an intradermal delivery device, forapplying said single variable domain or polypeptide to the skin.

DETAILED DESCRIPTION

Within this specification, the disclosure has been described, withreference to embodiments, in a way which enables a clear and concisespecification to be written. It is intended and should be appreciatedthat embodiments may be variously combined or separated without partingfrom the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, nucleic acidchemistry, hybridization techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods (seegenerally, Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.and Ausubel, et al., Short Protocols in Molecular Biology (1999) 4th Ed,John Wiley & Sons, Inc., which are incorporated herein by reference) andchemical methods.

Immunoglobulin: As used herein, “immunoglobulin” refers to a family ofpolypeptides which retain the immunoglobulin fold characteristic ofantibody molecules, which contain two β sheets and, usually, a conserveddisulphide bond.

Domain: As used herein “domain” refers to a folded protein structurewhich retains its tertiary structure independently of the rest of theprotein. Generally, domains are responsible for discrete functionalproperties of proteins and in many cases may be added, removed ortransferred to other proteins without loss of function of the remainderof the protein and/or of the domain. By single antibody variable domainor immunoglobulin single variable domain is meant a folded polypeptidedomain comprising sequences characteristic of antibody variable domains.It therefore includes complete antibody variable domains and modifiedvariable domains, for example in which one or more loops have beenreplaced by sequences which are not characteristic of antibody variabledomains, or antibody variable domains which have been truncated orcomprise N- or C-terminal extensions, as well as folded fragments ofvariable domains which retain at least in part the binding activity andspecificity of the full-length domain.

Immunoglobulin single variable domain: The phrase “immunoglobulin singlevariable domain” refers to an antibody variable domain (V_(H), V_(HH),V_(L)) or binding domain that specifically binds an antigen or epitopeindependently of different or other V regions or domains i.e. ismonovalent. An immunoglobulin single variable domain can be present in aformat (e.g., homo- or hetero-multimer) with other variable regions orvariable domains where the other regions or domains are not required forantigen binding by the single immunoglobulin variable domain (i.e.,where the immunoglobulin single variable domain binds antigenindependently of the additional variable domains). A “domain antibody”or “dAb” is an “immunoglobulin single variable domain” as the term isused herein. A “single antibody variable domain” or an “antibody singlevariable domain” is the same as an “immunoglobulin single variabledomain” as the term is used herein. An immunoglobulin single variabledomain is in one embodiment a human antibody variable domain, but alsoincludes single antibody variable domains from other species such asrodent (for example, as disclosed in WO 00/29004, the contents of whichare incorporated herein by reference in their entirety), nurse shark andCamelid VHH dAbs. Camelid VHH are immunoglobulin single variable domainpolypeptides that are derived from species including camel, llama,alpaca, dromedary, and guanaco, which produce heavy chain antibodiesnaturally devoid of light chains. The VHH may be humanized.

In all aspects of the disclosure, the immunoglobulin single variabledomain is independently selected from antibody heavy chain and lightchain single variable domains, e.g. V_(H), V_(L) and V_(HH).

As used herein an “antibody” refers to IgG, IgM, IgA, IgD or IgE or afragment (such as a Fab, F(ab′)₂, Fv, disulphide linked Fv, scFv, closedconformation multispecific antibody, disulphide-linked scFv, diabody)whether derived from any species naturally producing an antibody, orcreated by recombinant DNA technology; whether isolated from, forexample, serum, B-cells, hybridomas, transfectomas, yeast or bacteria.

Antibody format: In one embodiment, the immunoglobulin single variabledomain, polypeptide or ligand in accordance with the disclosure can beprovided in any antibody format. As used herein, “antibody format”refers to any suitable polypeptide structure in which one or moreantibody variable domains can be incorporated so as to confer bindingspecificity for antigen on the structure. A variety of suitable antibodyformats are known in the art, such as, chimeric antibodies, humanizedantibodies, human antibodies, single chain antibodies, bispecificantibodies, antibody heavy chains, antibody light chains, homodimers andheterodimers of antibody heavy chains and/or light chains,antigen-binding fragments of any of the foregoing (e.g., a Fv fragment(e.g., single chain Fv (scFv), a disulfide bonded Fv), a Fab fragment, aFab′ fragment, a F(ab′)₂ fragment), a single antibody variable domain(e.g., a dAb, V_(H), V_(HH), V_(L), and modified versions of any of theforegoing (e.g., modified by the covalent attachment of polyethyleneglycol or other suitable polymer or a humanized V_(HH)). In oneembodiment, alternative antibody formats include alternative scaffoldsin which the CDRs of any molecules in accordance with the disclosure canbe grafted onto a suitable protein scaffold or skeleton, such as anaffibody, a SpA scaffold, an LDL receptor class A domain, an avimer(see, e.g., U.S. Patent Application Publication Nos. 2005/0053973,2005/0089932, 2005/0164301) or an EGF domain. Further, the ligand can bebivalent (heterobivalent) or multivalent (heteromultivalent) asdescribed herein. In other embodiments, a “Universal framework” may beused wherein “Universal framework” refers to a single antibody frameworksequence corresponding to the regions of an antibody conserved insequence as defined by Kabat (“Sequences of Proteins of ImmunologicalInterest”, US Department of Health and Human Services) or correspondingto the human germline immunoglobulin repertoire or structure as definedby Chothia and Lesk, (1987) J. Mol. Biol. 196:910-917. The disclosureprovides for the use of a single framework, or a set of such frameworks,which has been found to permit the derivation of virtually any bindingspecificity through variation in the hypervariable regions alone.

In embodiments of the disclosure described throughout this disclosure,instead of the use of an anti-TGFbetaRII “dAb” in a peptide or ligand ofthe disclosure, it is contemplated that one of ordinary skill in the artcan use a polypeptide or domain that comprises one or more or all 3 ofthe CDRs of a dAb of the disclosure that binds TGFbetaRII (e.g., CDRsgrafted onto a suitable protein scaffold or skeleton, e.g. an affibody,an SpA scaffold, an LDL receptor class A domain or an EGF domain). Thedisclosure as a whole is to be construed accordingly to providedisclosure of polypeptides using such domains in place of a dAb. In thisrespect, see WO2008096158, the disclosure of which is incorporated byreference.

In one embodiment, the anti-TGFbetaRII immunoglobulin single variabledomain is any suitable immunoglobulin variable domain, and optionally isa human variable domain or a variable domain that comprises or isderived from a human framework region (e.g., DP47 or DPK9 frameworkregions).

Antigen: As described herein an “antigen” is a molecule that is bound bya binding domain according to the present disclosure. Typically,antigens are bound by antibody ligands and are capable of raising anantibody response in vivo. It may be, for example, a polypeptide,protein, nucleic acid or other molecule.

Epitope: An “epitope” is a unit of structure conventionally bound by animmunoglobulin V_(H)/V_(L) pair. Epitopes define the minimum bindingsite for an antibody, and thus represent the target of specificity of anantibody. In the case of a single domain antibody, an epitope representsthe unit of structure bound by a variable domain in isolation.

Binding: Typically, specific binding is indicated by a dissociationconstant (Kd) of 50 nanomolar or less, optionally 250 picomolar or less.Specific binding of an antigen-binding protein to an antigen or epitopecan be determined by a suitable assay, including, for example, Scatchardanalysis and/or competitive binding assays, such as radioimmunoassays(RIA), enzyme immunoassays such as ELISA and sandwich competitionassays, and the different variants thereof.

Binding affinity: Binding affinity is optionally determined usingsurface plasmon resonance (SPR) and BIACORE™ (Karlsson et al., 1991),using a BIACORE™ system (Uppsala, Sweden). The BIACORE™ system usessurface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect.23:1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) tomonitor biomolecular interactions in real time, and uses surface plasmonresonance which can detect changes in the resonance angle of light atthe surface of a thin gold film on a glass support as a result ofchanges in the refractive index of the surface up to 300 nm away.BIACORE™ analysis conveniently generates association rate constants,dissociation rate constants, equilibrium dissociation constants, andaffinity constants. Binding affinity is obtained by assessing theassociation and dissociation rate constants using a BIACORE™ surfaceplasmon resonance system (BIACORE™, Inc.). A biosensor chip is activatedfor covalent coupling of the target according to the manufacturer's(BIACORE™) instructions. The target is then diluted and injected overthe chip to obtain a signal in response units of immobilized material.Since the signal in resonance units (RU) is proportional to the mass ofimmobilized material, this represents a range of immobilized targetdensities on the matrix. Dissociation data are fit to a one-site modelto obtain k_(off)+/−s.d. (standard deviation of measurements).Pseudo-first order rate constant (Kd's) are calculated for eachassociation curve, and plotted as a function of protein concentration toobtain k_(on)+/−s.e. (standard error of fit). Equilibrium dissociationconstants for binding, Kd's, are calculated from SPR measurements ask_(off)/k_(on).

Another aspect of the disclosure provides an anti-TGFbetaRIIimmunoglobulin single variable domain that specifically binds to humanTGFbetaRII. In one embodiment, the variable domain binds humanTGFbetaRII with an equilibrium dissociation constant (KD) of about 50nM, 40 nM, 30 nM, 20 nM, 10 nM or less, optionally about 9, 8, 7, 6 or 5nM or less, optionally about 4 nM or less, about 3 nM or less or about 2nM or less or about 1 nM or less, optionally about 500 pM or less.Suitably, where the variable domain has an equilibrium dissociationconstant in the range of about 50 nM to 500 pM, it is particularlysuitable for local administration to a tissue of interest such as thelung. In this embodiment, a high concentration of such a “moderateaffinity” binder can be provided as an effective therapeutic. In anotherembodiment, the variable domain binds human TGFbetaRII with anequilibrium dissociation constant (KD) of about 500 pM or less,optionally about 450 pM, 400 pM, 350 pM, 300 pM, 250 pM, 200 pM, 150 pM,100 pM, 50 pM or less, optionally about 40 pM, 30 pM, 20 pM, 10 pM orless. Suitably, where the variable domain has a dissociation constant inthe range of about 500 pM to 10 pM, it is particularly suitable forsystemic administration such that the amount in any one tissue ofinterest is sufficient to provide an effective therapy. In thisembodiment, a low concentration of such a “high affinity” binder can beprovided as an effective therapeutic.

In one embodiment, single variable domains of the present disclosureshow cross-reactivity between human TGFbetaRII and TGFbetaRII fromanother species, such as mouse TGFbetaRII. In this embodiment, thevariable domains specifically bind human and mouse TGFbetaRII. This isparticularly useful, since drug development typically requires testingof lead drug candidates in mouse systems before the drug is tested inhumans. The provision of a drug that can bind human and mouse speciesallows one to test results in these system and make side-by-sidecomparisons of data using the same drug. This avoids the complication ofneeding to find a drug that works against a mouse TGFbetaRII and aseparate drug that works against human TGFbetaRII, and also avoids theneed to compare results in humans and mice using non-identical drugs.Cross reactivity between other species used in disease models such asdog or monkey such as cynomolgus monkey is also envisaged.

Optionally, the binding affinity of the immunoglobulin single variabledomain for at least mouse TGFbetaRII and the binding affinity for humanTGFbetaRII differ by no more than a factor of 10, 50 or 100.

CDRs: The immunoglobulin single variable domains (dAbs) described hereincontain complementarity determining regions (CDR1, CDR2 and CDR3). Thelocations of CDRs and frame work (FR) regions and a numbering systemhave been defined by Kabat et al. (Kabat, E. A. et al., Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, U.S. Government Printing Office (1991)). Theamino acid sequences of the CDRs (CDR1, CDR2, CDR3) of the V_(H) (CDRH1etc.) and V_(L) (CDRL1 etc.) (Vκ) dAbs disclosed herein will be readilyapparent to the person of skill in the art based on the well known Kabatamino acid numbering system and definition of the CDRs. According to theKabat numbering system, the most commonly used method based on sequencevariability, heavy chain CDR-H3 have varying lengths, insertions arenumbered between residue H100 and H101 with letters up to K (i.e. H100,H100A . . . H100K, H101). CDRs can alternatively be determined using thesystem of Chothia (based on location of the structural loop regions)(Chothia et al., (1989) Conformations of immunoglobulin hypervariableregions; Nature 342, p877-883), according to AbM (compromise betweenKabat and Chothia) or according to the Contact method (based on crystalstructures and prediction of contact residues with antigen) as follows.See http://www.bioinf.org.uk/abs/for suitable methods for determiningCDRs.

Once each residue has been numbered, one can then apply the followingCDR definitions:

Kabat: CDR H1: 31-35/35A/35B CDR H2: 50-65  CDR H3: 95-102 CDR L1: 24-34CDR L2: 50-56 CDR L3: 89-97 Chothia: CDR H1: 26-32 CDR H2: 52-56  CDRH3: 95-102 CDR L1: 24-34 CDR L2: 50-56 CDR L3: 89-97 AbM: (using Kabatnumbering): (using Chothia numbering): CDR H1: 26-35/35A/35B 26-35 CDRH2: 50-58 —  CDR H3: 95-102 — CDR L1: 24-34 — CDR L2: 50-56 — CDR L3:89-97 — Contact: (using Kabat numbering): (using Chothia numbering): CDRH1: 30-35/35A/35B 30-35 CDR H2: 47-58 —  CDR H3: 93-101 — CDR L1: 30-36— CDR L2: 46-55 — CDR L3: 89-96 — (″—″ means the same numbering asKabat)

Accordingly, a person skilled in the art is able to deduce from a givensingle variable domain sequence, e.g. one having a sequence as set outin any one of SEQ ID NO:1-38, 204, 206, 208, 214, 234, 236, 238, 240,263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287 whichCDR sequences are contained within them using the various methodsoutlined herein. For example, for a given single variable domainsequence e.g. SEQ ID NO:1 a skilled person is able to determine theCDR1, CDR2 and CDR3 sequences contained therein using any one or acombination of the CDR definition methods mentioned above. When usingthe Kabat CDR definition, the skilled person is able to determine thatCDR1, CDR2 and CDR 3 sequences are those set forth in SEQ ID NO:77, 113and 149 respectively. Suitably, CDR sequences are determined using themethod of Kabat described herein. In one embodiment, the CDR sequencesof each sequence are those set out in tables 1, 2, 9 and 13. In anembodiment a CDR1 sequence is a CDR1 sequence selected from SEQ IDNO:77-112, 241, 244, 247, and 250. In an embodiment a CDR2 sequence is aCDR2 sequence selected from SEQ ID NO:113-148, 242, 245, 248, and 251.In an embodiment a CDR3 sequence is a CDR3 sequence selected from SEQ IDNO:149-184, 243, 246, 249, and 252.

A CDR variant or variant binding unit includes an amino acid sequencemodified by at least one amino acid, wherein said modification can bechemical or a partial alteration of the amino acid sequence (for exampleby no more than 10 amino acids), which modification permits the variantto retain the biological characteristics of the unmodified sequence. Forexample, the variant is a functional variant which specifically binds toTGFbetaRII. A partial alteration of the CDR amino acid sequence may beby deletion or substitution of one to several amino acids, or byaddition or insertion of one to several amino acids, or by a combinationthereof (for example by no more than 10 amino acids). The CDR variant orbinding unit variant may contain 1, 2, 3, 4, 5 or 6 amino acidsubstitutions, additions or deletions, in any combination, in the aminoacid sequence. The CDR variant or binding unit variant may contain 1, 2or 3 amino acid substitutions, insertions or deletions, in anycombination, in the amino acid sequence. The substitutions in amino acidresidues may be conservative substitutions, for example, substitutingone hydrophobic amino acid for an alternative hydrophobic amino acid.For example leucine may be substituted with valine, or isoleucine.

TGFbetaRII: As used herein “TGFbetaRII” (transforming growth factor betatype II receptor; TGFβRII) refers to naturally occurring or endogenousmammalian TGFbetaRII proteins and to proteins having an amino acidsequence which is the same as that of a naturally occurring orendogenous corresponding mammalian TGFbetaRII protein (e.g., recombinantproteins, synthetic proteins (i.e., produced using the methods ofsynthetic organic chemistry)). Accordingly, as defined herein, the termincludes mature TGFbetaRII protein, polymorphic or allelic variants, andother isoforms of TGFbetaRII and modified or unmodified forms of theforegoing (e.g., lipidated, glycosylated). Naturally occurring orendogenous TGFbetaRII includes wild type proteins such as matureTGFbetaRII, polymorphic or allelic variants and other isoforms andmutant forms which occur naturally in mammals (e.g., humans, non-humanprimates). Such proteins can be recovered or isolated from a sourcewhich naturally expresses TGFbetaRII, for example. These proteins andproteins having the same amino acid sequence as a naturally occurring orendogenous corresponding TGFbetaRII, are referred to by the name of thecorresponding mammal. For example, where the corresponding mammal is ahuman, the protein is designated as a human TGFbetaRII. Human TGFbetaRIIis described, for example, by Lin, et al., Cell 1992, Vol. 68(4), p.775-785 and GenBank Accession No. M85079.

Human TGFbetaRII is a transmembrane receptor consisting of 567 aminoacids with an extracellular domain of approximately 159 amino acids, atransmembrane domain and a cytoplasmic domain which comprises a proteinkinase domain for signal transduction.

As used herein “TGFbetaRII” also includes a portion or fragment ofTGFbetaRII. In one embodiment, such a portion or fragment includes theextracellular domain of TGFbetaRII or a portion thereof.

By “anti-TGFbetaRII” with reference to an immunoglobulin single variabledomain, polypeptide, ligand, fusion protein or so forth is meant amoiety which recognises and binds TGFbetaRII. In one embodiment an“anti-TGFbetaRII” specifically recognises and/or specifically binds tothe protein TGFbetaRII, and, suitably, human TGFbetaRII. In anotherembodiment, the anti-TGFbetaRII immunoglobulin single variable domain inaccordance with the disclosure also binds to mouse TGFbetaRII (GenBankaccession number NM_(—)029575; described, for example in Massague etal., Cell 69 (7), 1067-1070 (1992)).

“TGFbeta” includes isoforms such as TGFbeta1, TGFbeta2 and TGFbeta3.

TGFbeta binds TGFbetaRII and, in a complex with TGFbetaRI initiates asignaling pathway. Accordingly, TGFbeta activity and inhibition orneutralization of TGFbeta activity can be determined through any assaywhich measures an output of TGFbeta signaling. TGFbeta signaling isreviewed, for example in Itoh, et al., Eur. J. Biochem 2000, Vol. 267,p. 6954; Dennler, et al., Journal of Leucocyte Biol. 2002, 71(5), p.731-40. Thus, TGFbeta activity can be tested in a number of differentassays familiar to the person skilled in the art. “Inhibition” or“Neutralization” means that a biological activity of TGFbeta is reducedeither totally or partially in the presence of the immunoglobulin singlevariable domain of the present disclosure in comparison to the activityof TGFbeta in the absence of such immunoglobulin single variable domain.

In one embodiment, an inhibition or neutralisation of TGFbeta activityis tested in an IL-11 release assay. In this embodiment, the ability ofthe immunoglobulin single variable domain in accordance with thedisclosure is tested for its ability to inhibit human TGFbeta1(TGFbeta1; TGF-β1) stimulated IL-11 release from cells such as A549cells. TGFbeta1 (TGF-β1) binds directly to TGFbetaRII (TGF-βRII) andinduces the assembly of the TGFbetaRI/RII (TGF-βRI/II) complex.TGFbetaRI (TGF-βRI) is phosphorylated and is able to signal throughseveral pathways including the Smad 4 pathway. Activation of the Smad 4pathway results in the release of IL-11. The IL-11 is secreted into thecell supernatant and is then measured by colourmetric ELISA. SuitableIL-11 release assays are described herein, such as the Human IL-11Quantikine ELISA assay kit supplied by R & D systems (ref. D1100).

In another embodiment, TGFbeta activity is tested in an assay for theability of the immunoglobulin single variable domain in accordance withthe disclosure to inhibit TGFbeta-induced expression of CAGA-luciferasein MC3T3-E1 cells in a MC3T3-E1 luciferase assay. Three copies of aTGFbeta-responsive sequence motif, termed a CAGA box are present in thehuman PAI-1 promoter and specifically binds Smad3 and 4 proteins.Cloning multiple copies of the CAGA box into a luciferase reporterconstruct confers TGFbeta responsiveness to cells transfected with thereporter system. One suitable assay is described herein and usesMC3T3-E1 cells (mouse osteoblasts) stably transfected with a[CAGA]₁₂-luciferase reporter construct (Dennler, et al., (1998) EMBO J.17, 3091-3100).

Other suitable assays include a human SBE beta-lactamase cell assay(INVITROGEN®, cell sensor assay). Examples of suitable assays aredescribed herein.

Suitably, the immunoglobulin single variable domain, polypeptide, ligandor fusion protein in accordance with the disclosure does not, itselfactivate TGFbetaRII receptor signalling. Accordingly, in one embodiment,the immunoglobulin single variable domain, polypeptide, ligand or fusionprotein in accordance with the disclosure is devoid of agonist activityat 10 μM. Agonist activity can be determined by testing a compound ofinterest in a TGFbetaRII assay as described herein in the absence ofTGFbeta. Where TGFbeta is absent, agonist activity of a compound ofinterest would be detected by detecting TGFbetaRII signalling.

Homology: Sequences similar or homologous (e.g., at least about 70%sequence identity) to the sequences disclosed herein are also part ofthe disclosure. In some embodiments, the sequence identity at the aminoacid level can be about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or higher. At the nucleic acid level, the sequenceidentity can be about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively, substantialidentity exists when the nucleic acid segments will hybridize underselective hybridization conditions (e.g., very high stringencyhybridization conditions), to the complement of the strand. The nucleicacids may be present in whole cells, in a cell lysate, or in a partiallypurified or substantially pure form.

As used herein, the terms “low stringency,” “medium stringency,” “highstringency,” or “very high stringency” conditions describe conditionsfor nucleic acid hybridization and washing. Guidance for performinghybridization reactions can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated herein by reference in its entirety. Aqueous and nonaqueousmethods are described in that reference and either can be used. Specifichybridization conditions referred to herein are as follows: (1) lowstringency hybridization conditions in 6× sodium chloride/sodium citrate(SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS atleast at 50° C. (the temperature of the washes can be increased to 55°C. for low stringency conditions); (2) medium stringency hybridizationconditions in 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 60° C.; (3) high stringency hybridizationconditions in 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 65° C.; and optionally (4) very high stringencyhybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C.,followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very highstringency conditions (4) are the preferred conditions and the ones thatshould be used unless otherwise specified.

Calculations of “homology” or “sequence identity” or “similarity”between two sequences (the terms are used interchangeably herein) areperformed as follows. The sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Inone embodiment, the length of a reference sequence aligned forcomparison purposes is at least about 30%, optionally at least about40%, optionally at least about 50%, optionally at least about 60%, andoptionally at least about 70%, 80%, 85% 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% of the length of the reference sequence. Theamino acid residues or nucleotides at corresponding amino acid positionsor nucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“homology” is equivalent to amino acid or nucleic acid “identity”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

Amino acid and nucleotide sequence alignments and homology, similarityor identity, as defined herein are optionally prepared and determinedusing the algorithm BLAST 2 Sequences, using default parameters(Tatusova, T. A. et al., FEMS Microbiol Lett, 174:187-188 (1999)).Alternatively, the BLAST algorithm (version 2.0) is employed forsequence alignment, with parameters set to default values. BLAST (BasicLocal Alignment Search Tool) is the heuristic search algorithm employedby the programs blastp, blastn, blastx, tblastn, and tblastx; theseprograms ascribe significance to their findings using the statisticalmethods of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA87(6):2264-8.

Ligand: As used herein, the term “ligand” refers to a compound thatcomprises at least one peptide, polypeptide or protein moiety that has abinding site with binding specificity for TGFbetaRII. A ligand can alsobe referred to as a “binding moiety”.

The ligands or binding moieties according to the disclosure optionallycomprise immunoglobulin variable domains which have different bindingspecificities, and do not contain variable domain pairs which togetherform a binding site for target compound (i.e., do not comprise animmunoglobulin heavy chain variable domain and an immunoglobulin lightchain variable domain that together form a binding site for TGFbetaRII).Optionally, each domain which has a binding site that has bindingspecificity for a target is an immunoglobulin single variable domain(e.g., immunoglobulin single heavy chain variable domain (e.g., V_(H),V_(HH)), immunoglobulin single light chain variable domain (e.g.,V_(L))) that has binding specificity for a desired target (e.g.,TGFbetaRII).

Thus, “ligands” include polypeptides that comprise two or moreimmunoglobulin single variable domains wherein each immunoglobulinsingle variable domain binds to a different target. Ligands also includepolypeptides that comprise at least two immunoglobulin single variabledomains or the CDR sequences of the single variable domains that binddifferent targets in a suitable format, such as an antibody format(e.g., IgG-like format, scFv, Fab, Fab′, F(ab′)₂) or a suitable proteinscaffold or skeleton, such as an affibody, a SpA scaffold, an LDLreceptor class A domain, an EGF domain, avimer and dual- andmulti-specific ligands as described herein.

The polypeptide domain which has a binding site that has bindingspecificity for a target (e.g., TGFbetaRII) can also be a protein domaincomprising a binding site for a desired target, e.g., a protein domainis selected from an affibody, a SpA domain, an LDL receptor class Adomain, an avimer (see, e.g., U.S. Patent Application Publication Nos.2005/0053973, 2005/0089932, 2005/0164301). If desired, a “ligand” canfurther comprise one or more additional moieties that can eachindependently be a peptide, polypeptide or protein moiety or anon-peptidic moiety (e.g., a polyalkylene glycol, a lipid, acarbohydrate). For example, the ligand can further comprise a half-lifeextending moiety as described herein (e.g., a polyalkylene glycolmoiety, a moiety comprising albumin, an albumin fragment or albuminvariant, a moiety comprising transferrin, a transferrin fragment ortransferrin variant, a moiety that binds albumin, a moiety that bindsneonatal Fc receptor).

Competes: As referred to herein, the term “competes” means that thebinding of a first target (e.g., TGFbetaRII) to its cognate targetbinding domain (e.g., immunoglobulin single variable domain) isinhibited in the presence of a second binding domain (e.g.,immunoglobulin single variable domain) that is specific for said cognatetarget. For example, binding may be inhibited sterically, for example byphysical blocking of a binding domain or by alteration of the structureor environment of a binding domain such that its affinity or avidity fora target is reduced. See WO2006038027 for details of how to performcompetition ELISA and competition BIACORE™ experiments to determinecompetition between first and second binding domains, the details ofwhich are incorporated herein by reference to provide explicitdisclosure for use in the present disclosure. The disclosure includesantigen binding proteins, specifically single variable domains,polypeptides, ligands and fusion proteins, that compete with any one ofsingle variable domains of SEQ ID NO:1-38. In a particular embodimentthere is provided a TGFbetaRII binding protein which competes with anyone of single variable domains of SEQ ID NO:1-38 and also has a KD of 50nM or less to TGFbetaRII. In a particular embodiment the KD is between10 pM and 50 nM. In a particular embodiment, the KD is between 10 pM and10 nM. In a particular embodiment, the KD is between 100 pM and 10 nM.In a particular embodiment the KD is approximately 100 pM.

TGFbeta signaling: Suitably, the single variable domain, polypeptide orligand of the disclosure can neutralize TGFbeta signaling throughTGFbetaRII. By “neutralizing”, it is meant that the normal signalingeffect of TGFbeta is blocked such that the presence of TGFbeta has aneutral effect on TGFbetaRII signaling. Suitable methods for measuring aneutralizing effect include assays for TGFbeta signaling as describedherein. In one embodiment, neutralization is observed as a % inhibitionof TFGbeta activity in a TGFbeta signaling assay. In one embodiment, thesingle variable domain or polypeptide binds to the extracellular domainof TGFbetaRII thereby inhibiting/blocking the binding of TGFbeta to theextracellular domain of TGFbetaRII. Suitably, the single variable domainor polypeptide is useful where there is an excess of bioavailableTGFbeta and the single variable domain or polypeptide serves to inhibitthe signaling activity of the bioavailable TGFbeta through inhibitingbinding or TGFbeta to its cognate receptor TGFbetaRII.

As used herein, the term “antagonist of TGFbetaRII” or “anti-TGFbetaRIIantagonist” or the like refers to an agent (e.g., a molecule, acompound) which binds TGFbetaRII and can inhibit a (i.e., one or more)function of TGFbetaRII. For example, an antagonist of TGFbetaRII caninhibit the binding of TGFbeta to TGFbetaRII and/or inhibit signaltransduction mediated through TGFbetaRII. Accordingly, TGFbeta-mediatedprocesses and cellular responses can be inhibited with an antagonist ofTGFbetaRII.

In one embodiment, the ligand (e.g., immunoglobulin single variabledomain) that binds TGFbetaRII inhibits binding of TGFbeta to aTGFbetaRII receptor with an inhibitory concentration 50 (IC50) that is≦about 10 μM, ≦about 1 μM, ≦about 100 nM, ≦about 50 nM, ≦about 10 nM,≦about 5 nM, ≦about 1 nM, ≦about 500 pM, ≦about 300 pM, ≦about 100 pM,or ≦about 10 pM. In a particular embodiment, an anti-TGFbetaRIIimmunoglobulin single variable domain of the disclosure has an IC50 of15 μM or less. The IC50 is optionally determined using an in vitroTGFbeta receptor binding assay, or cell assay, such as the assaydescribed herein.

It is also contemplated that the ligand (e.g., immunoglobulin singlevariable domain) optionally inhibit TGFbetaRII induced functions in asuitable in vitro assay with a neutralizing dose 50 (ND50) that is≦about 10 μM, ≦about 1 μM, ≦about 100 nM, ≦about 50 nM, ≦about 10 nM,≦about 5 nM, ≦about 1 nM, ≦about 500 pM, ≦about 300 pM, ≦about 100 pM,≦about 10 pM, ≦about 1 pM about 500 fM, ≦about 300 fM, ≦about 100 fM,≦about 10 fM. In a particular embodiment, an anti-TGFbetaRIIimmunoglobulin single variable domain of the disclosure achieves greaterthan 40% neutralisation of TGF-β.

“dual-specific ligand”: In one embodiment, the immunoglobulin singlevariable domain, polypeptide or ligand in accordance with the disclosurecan be part of a “dual-specific ligand” which refers to a ligandcomprising a first antigen or epitope binding site (e.g., firstimmunoglobulin single variable domain) and a second antigen or epitopebinding site (e.g., second immunoglobulin single variable domain),wherein the binding sites or variable domains are capable of binding totwo antigens (e.g., different antigens or two copies of the sameantigen) or two epitopes on the same antigen which are not normallybound by a monospecific immunoglobulin. For example, the two epitopesmay be on the same antigen, but are not the same epitope or sufficientlyadjacent to be bound by a monospecific ligand. In one embodiment,dual-specific ligands according to the disclosure are composed ofbinding sites or variable domains which have different specificities,and do not contain mutually complementary variable domain pairs (i.e.V_(H)/V_(L) pairs) which have the same specificity (i.e., do not form aunitary binding site).

In one embodiment, a “dual-specific ligand” may bind to TGFbetaRII andto another target molecule. For example, another target molecule may bea tissue-specific target molecule such that the dual-specific ligand ofthe disclosure enables an anti-TGFbetaRII polypeptide or immunoglobulinsingle variable domain in accordance with the disclosure to be targetedto a tissue of interest. Such tissues include lung, liver and so forth.

Multispecific dAb multimers are also provided. This includes a dAbmultimer comprising an anti-TGFbetaRII immunoglobulin single variabledomain according to any aspect of the disclosure and one or more singlevariable domains each of which binds to a different target (e.g. atarget other than TGFbetaRII). In an embodiment a bispecific dAbmultimer is provided e.g. a dab multimer comprising one or moreanti-TGFbetaRII immunoglobulin single variable domains according to anyaspect of the disclosure and one or more dabs which bind to a second,different target. In an embodiment a trispecific dAb multimer isprovided.

The ligands of the disclosure (e.g., polypeptides, dAbs and antagonists)can be formatted as a fusion protein that contains a firstimmunoglobulin single variable domain that is fused directly to a secondimmunoglobulin single variable domain. If desired such a format canfurther comprise a half-life extending moiety. For example, the ligandcan comprise a first immunoglobulin single variable domain that is fuseddirectly to a second immunoglobulin single variable domain that is fuseddirectly to an immunoglobulin single variable domain that binds serumalbumin.

Generally, the orientation of the polypeptide domains that have abinding site with binding specificity for a target, and whether theligand comprises a linker, is a matter of design choice. However, someorientations, with or without linkers, may provide better bindingcharacteristics than other orientations. All orientations (e.g.,dAb1-linker-dAb2; dAb2-linker-dAb1) are encompassed by the disclosureare ligands that contain an orientation that provides desired bindingcharacteristics can be easily identified by screening.

Polypeptides and dAbs according to the disclosure, including dAbmonomers, dimers and trimers, can be linked to an antibody Fc region,comprising one or both of C_(H)2 and C_(H)3 domains, and optionally ahinge region. For example, vectors encoding ligands linked as a singlenucleotide sequence to an Fc region may be used to prepare suchpolypeptides. In an embodiment there is provided a dAb-Fc fusion.

The disclosure moreover provides dimers, trimers and polymers of theaforementioned dAb monomers.

Target: As used herein, the phrase “target” refers to a biologicalmolecule (e.g., peptide, polypeptide, protein, lipid, carbohydrate) towhich a polypeptide domain which has a binding site can bind. The targetcan be, for example, an intracellular target (e.g., an intracellularprotein target), a soluble target (e.g., a secreted), or a cell surfacetarget (e.g., a membrane protein, a receptor protein). In oneembodiment, the target is TGFbetaRII. In another embodiment, the targetis TGFbetaRII extracellular domain.

Complementary: As used herein “complementary” refers to when twoimmunoglobulin domains belong to families of structures which formcognate pairs or groups or are derived from such families and retainthis feature. For example, a V_(H) domain and a V_(L) domain of anantibody are complementary; two V_(H) domains are not complementary, andtwo V_(L) domains are not complementary. Complementary domains may befound in other members of the immunoglobulin superfamily, such as the Vαand Vβ (or γ and δ) domains of the T-cell receptor. Domains which areartificial, such as domains based on protein scaffolds which do not bindepitopes unless engineered to do so, are non-complementary. Likewise,two domains based on (for example) an immunoglobulin domain and afibronectin domain are not complementary.

“Affinity” and “avidity” are terms of art that describe the strength ofa binding interaction. With respect to the ligands of the disclosure,avidity refers to the overall strength of binding between the targets(e.g., first target and second target) on the cell and the ligand.Avidity is more than the sum of the individual affinities for theindividual targets.

Nucleic acid molecules, vectors and host cells: The disclosure alsoprovides isolated and/or recombinant nucleic acid molecules encodingligands (single variable domains, fusion proteins, polypeptides,dual-specific ligands and multispecific ligands) as described herein.

Nucleic acids referred to herein as “isolated” are nucleic acids whichhave been separated away from the nucleic acids of the genomic DNA orcellular RNA of their source of origin (e.g., as it exists in cells orin a mixture of nucleic acids such as a library), and include nucleicacids obtained by methods described herein or other suitable methods,including essentially pure nucleic acids, nucleic acids produced bychemical synthesis, by combinations of biological and chemical methods,and recombinant nucleic acids which are isolated (see e.g., Daugherty,B. L. et al., Nucleic Acids Res., 19(9): 2471 2476 (1991); Lewis, A. P.and J. S. Crowe, Gene, 101: 297-302 (1991)).

Nucleic acids referred to herein as “recombinant” are nucleic acidswhich have been produced by recombinant DNA methodology, including thosenucleic acids that are generated by procedures which rely upon a methodof artificial recombination, such as the polymerase chain reaction (PCR)and/or cloning into a vector using restriction enzymes.

In certain embodiments, the isolated and/or recombinant nucleic acidcomprises a nucleotide sequence encoding an immunoglobulin singlevariable domain, polypeptide or ligand, as described herein, whereinsaid ligand comprises an amino acid sequence that has at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% amino acid sequence identity with the amino acidsequence of a dAb that binds TGFbetaRII disclosed herein, e.g. aminoacid sequences set out in any of SEQ ID NOS: 1-38. Nucleotide sequenceidentity can be determined over the whole length of the nucleotidesequence that encodes the selected anti-TGFbetaRII dAb. In an embodimentthe nucleic acid sequence comprises or consists of a nucleic acidsequence at least 80% identical to of any one of SEQ ID NO:39-66. In anembodiment the nucleic acid sequence comprises or consists of a nucleicacid sequence of any one of SEQ ID NO:39-76.

Embodiments of the disclosure also provide codon optimized nucleotidesequences encoding polypeptides and variable domains as disclosed hereine.g. optimised for expression in bacterial, mammalian or yeast cells.

The disclosure also provides a vector comprising a recombinant nucleicacid molecule of the disclosure. In certain embodiments, the vector isan expression vector comprising one or more expression control elementsor sequences that are operably linked to the recombinant nucleic acid ofthe disclosure. The disclosure also provides a recombinant host cellcomprising a recombinant nucleic acid molecule or vector of thedisclosure. Suitable vectors (e.g., plasmids, phagemids), expressioncontrol elements, host cells and methods for producing recombinant hostcells of the disclosure are well-known in the art, and examples arefurther described herein.

Suitable expression vectors can contain a number of components, forexample, an origin of replication, a selectable marker gene, one or moreexpression control elements, such as a transcription control element(e.g., promoter, enhancer, terminator) and/or one or more translationsignals, a signal sequence or leader sequence, and the like. Expressioncontrol elements and a signal sequence, if present, can be provided bythe vector or other source. For example, the transcriptional and/ortranslational control sequences of a cloned nucleic acid encoding anantibody chain can be used to direct expression.

A promoter can be provided for expression in a desired host cell.Promoters can be constitutive or inducible. For example, a promoter canbe operably linked to a nucleic acid encoding an antibody, antibodychain or portion thereof, such that it directs transcription of thenucleic acid. A variety of suitable promoters for prokaryotic (e.g.,lac, tac, T3, T7 promoters for E. coli) and eukaryotic (e.g., SimianVirus 40 early or late promoter, Rous sarcoma virus long terminal repeatpromoter, cytomegalovirus promoter, adenovirus late promoter) hosts areavailable.

In addition, expression vectors typically comprise a selectable markerfor selection of host cells carrying the vector, and, in the case of areplicable expression vector, an origin of replication. Genes encodingproducts which confer antibiotic or drug resistance are commonselectable markers and may be used in prokaryotic (e.g., lactamase gene(ampicillin resistance), Tet gene for tetracycline resistance) andeukaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolicacid), ampicillin, or hygromycin resistance genes). Dihydrofolatereductase marker genes permit selection with methotrexate in a varietyof hosts. Genes encoding the gene product of auxotrophic markers of thehost (e.g., LEU2, URA3, HIS3) are often used as selectable markers inyeast. Use of viral (e.g., baculovirus) or phage vectors, and vectorswhich are capable of integrating into the genome of the host cell, suchas retroviral vectors, are also contemplated. Suitable expressionvectors for expression in mammalian cells and prokaryotic cells (E.coli), insect cells (Drosophila Schnieder S2 cells, Sf9) and yeast (P.methanolica, P. pastoris, S. cerevisiae) are well-known in the art.

Suitable host cells can be prokaryotic, including bacterial cells suchas E. coli, B. subtilis and/or other suitable bacteria; eukaryoticcells, such as fungal or yeast cells (e.g., Pichia pastoris, Aspergillussp., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurosporacrassa), or other lower eukaryotic cells, and cells of higher eukaryotessuch as those from insects (e.g., Drosophila Schnieder S2 cells, Sf9insect cells (WO 94/26087 (O'Connor)), mammals (e.g., COS cells, such asCOS-1 (ATCC Accession No. CRL-1650) and COS-7 (ATCC Accession No.CRL-1651), CHO (e.g., ATCC Accession No. CRL-9096, CHO DG44 (Urlaub, G.and Chasin, L A., Proc. Natl. Acad. Sci. USA, 77(7):4216-4220 (1980))),293 (ATCC Accession No. CRL-1573), HeLa (ATCC Accession No. CCL-2), CV1(ATCC Accession No. CCL-70), WOP (Dailey, L., et al., J. Virol.,54:739-749 (1985), 3T3, 293T (Pear, W. S., et al., Proc. Natl. Acad.Sci. U.S.A., 90:8392-8396 (1993)) NS0 cells, SP2/0, HuT 78 cells and thelike, or plants (e.g., tobacco). (See, for example, Ausubel, F. M. etal., eds. Current Protocols in Molecular Biology, Greene PublishingAssociates and John Wiley & Sons Inc. (1993). In some embodiments, thehost cell is an isolated host cell and is not part of a multicellularorganism (e.g., plant or animal). In certain embodiments, the host cellis a non-human host cell. The disclosure also provides a method forproducing a ligand (e.g., dual-specific ligand, multispecific ligand) ofthe disclosure, comprising maintaining a recombinant host cellcomprising a recombinant nucleic acid of the disclosure under conditionssuitable for expression of the recombinant nucleic acid, whereby therecombinant nucleic acid is expressed and a ligand is produced. In someembodiments, the method further comprises isolating the ligand.

Reference is made to WO200708515, page 161, line 24 to page 189, line 10for details of disclosure that is applicable to embodiments of thepresent disclosure. This disclosure is hereby incorporated herein byreference as though it appears explicitly in the text of the presentdisclosure and relates to the embodiments of the present disclosure, andto provide explicit support for disclosure to incorporated into claimsbelow. This includes disclosure presented in WO200708515, page 161, line24 to page 189, line 10 providing details of the “Preparation ofImmunoglobulin Based Ligands”, “Library vector systems”, “LibraryConstruction”, “Combining Single Variable Domains”, “Characterisation ofLigands”, “Structure of Ligands”, “Skeletons”, “Protein Scaffolds”,“Scaffolds for Use in Constructing Ligands”, “Diversification of theCanonical Sequence” and “Therapeutic and diagnostic compositions anduses”, as well as definitions of “operably linked”, “naive”,“prevention”, “suppression”, “treatment”, “allergic disease”,“Th2-mediated disease”, “therapeutically-effective dose” and“effective”.

The phrase, “half-life” refers to the time taken for the serumconcentration of the immunoglobulin single variable domain, polypeptideor ligand to reduce by 50%, in vivo, for example due to degradation ofthe ligand and/or clearance or sequestration of the ligand by naturalmechanisms. The ligands of the disclosure can be stabilized in vivo andtheir half-life increased by binding to molecules which resistdegradation and/or clearance or sequestration. Typically, such moleculesare naturally occurring proteins which themselves have a long half-lifein vivo. The half-life of a ligand is increased if its functionalactivity persists, in vivo, for a longer period than a similar ligandwhich is not specific for the half-life increasing molecule. Thus aligand specific for HSA and a target molecules is compared with the sameligand wherein the specificity to HSA is not present, that is does notbind HSA but binds another molecule. Typically, the half-life isincreased by 10%, 20%, 30%, 40%, 50% or more. Increases in the range of2×, 3×, 4×, 5×, 10×, 20×, 30×, 40×, 50× or more of the half-life arepossible. Alternatively, or in addition, increases in the range of up to30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, 150× of the half life arepossible.

Formats: Increased half-life can be useful in in vivo applications ofimmunoglobulins, especially antibodies and most especially antibodyfragments of small size. Such fragments (Fvs, disulphide bonded Fvs,Fabs, scFvs, dAbs) are generally rapidly cleared from the body. dAbs,polypeptides or ligands in accordance with the disclosure can be adaptedto provide increased half-life in vivo and consequently longerpersistence times in the body of the functional activity of the ligand.

Methods for pharmacokinetic analysis and determination of ligandhalf-life will be familiar to those skilled in the art. Details may befound in Kenneth, A et al: Chemical Stability of Pharmaceuticals: AHandbook for Pharmacists and in Peters et al, Pharmacokinetic analysis:A Practical Approach (1996). Reference is also made to“Pharmacokinetics”, M Gibaldi & D Perron, published by Marcel Dekker,2nd Rev. ex edition (1982), which describes pharmacokinetic parameterssuch as t alpha and t beta half lives and area under the curve (AUC).

Half lives (t½ alpha and t½ beta) and AUC can be determined from a curveof serum concentration of ligand against time. The WINNONLIN™ analysispackage (available from Pharsight Corp., Mountain View, Calif. 94040,USA) can be used, for example, to model the curve. In a first phase (thealpha phase) the ligand is undergoing mainly distribution in thepatient, with some elimination. A second phase (beta phase) is theterminal phase when the ligand has been distributed and the serumconcentration is decreasing as the ligand is cleared from the patient.The t alpha half life is the half life of the first phase and the t betahalf life is the half life of the second phase. Thus, in one embodiment,the present disclosure provides a ligand or a composition comprising aligand according to the disclosure having a tα halflife in the range of15 minutes or more. In one embodiment, the lower end of the range is 30minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6hours, 7 hours, 10 hours, 11 hours or 12 hours. In addition, oralternatively, a ligand or composition according to the disclosure willhave a to half life in the range of up to and including 12 hours. In oneembodiment, the upper end of the range is 11, 10, 9, 8, 7, 6 or 5 hours.An example of a suitable range is 1 to 6 hours, 2 to 5 hours or 3 to 4hours.

In one embodiment, the present disclosure provides a ligand(polypeptide, dAb or antagonist) or a composition comprising a ligandaccording to the disclosure having a tβ halflife in the range of about2.5 hours or more. In one embodiment, the lower end of the range isabout 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7hours, about 10 hours, about 11 hours, or about 12 hours. In addition,or alternatively, a ligand or composition according to the disclosurehas a tβ halflife in the range of up to and including 21 days. In oneembodiment, the upper end of the range is about 12 hours, about 24hours, about 2 days, about 3 days, about 5 days, about 10 days, about 15days or about 20 days. In one embodiment a ligand or compositionaccording to the disclosure will have a tβ half life in the range about12 to about 60 hours. In a further embodiment, it will be in the rangeabout 12 to about 48 hours. In a further embodiment still, it will be inthe range about 12 to about 26 hours.

In addition, or alternatively to the above criteria, the presentdisclosure provides a ligand or a composition comprising a ligandaccording to the disclosure having an AUC value (area under the curve)in the range of about 1 mg·min/ml or more. In one embodiment, the lowerend of the range is about 5, about 10, about 15, about 20, about 30,about 100, about 200 or about 300 mg·min/ml. In addition, oralternatively, a ligand or composition according to the disclosure hasan AUC in the range of up to about 600 mg·min/ml. In one embodiment, theupper end of the range is about 500, about 400, about 300, about 200,about 150, about 100, about 75 or about 50 mg·min/ml. In one embodimenta ligand according to the disclosure will have a AUC in the rangeselected from the group consisting of the following: about 15 to about150 mg·min/ml, about 15 to about 100 mg·min/ml, about 15 to about 75mg·min/ml, and about 15 to about 50 mg·min/ml.

Polypeptides and dAbs of the disclosure and antagonists comprising thesecan be formatted to have a larger hydrodynamic size, for example, byattachment of a PEG group, serum albumin, transferrin, transferrinreceptor or at least the transferrin-binding portion thereof, anantibody Fc region, or by conjugation to an antibody domain. Forexample, polypeptides dAbs and antagonists formatted as a largerantigen-binding fragment of an antibody or as an antibody (e.g.,formatted as a Fab, Fab′, F(ab)₂, F(ab′)₂, IgG, scFv).

As used herein, “hydrodynamic size” refers to the apparent size of amolecule (e.g., a protein molecule, ligand) based on the diffusion ofthe molecule through an aqueous solution. The diffusion or motion of aprotein through solution can be processed to derive an apparent size ofthe protein, where the size is given by the “Stokes radius” or“hydrodynamic radius” of the protein particle. The “hydrodynamic size”of a protein depends on both mass and shape (conformation), such thattwo proteins having the same molecular mass may have differinghydrodynamic sizes based on the overall conformation of the protein.

Hydrodynamic size of the ligands (e.g., dAb monomers and multimers) ofthe disclosure may be determined using methods which are well known inthe art. For example, gel filtration chromatography may be used todetermine the hydrodynamic size of a ligand. Suitable gel filtrationmatrices for determining the hydrodynamic sizes of ligands, such ascross-linked agarose matrices, are well known and readily available.

The size of a ligand format (e.g., the size of a PEG moiety attached toa dAb monomer), can be varied depending on the desired application. Forexample, where ligand is intended to leave the circulation and enterinto peripheral tissues, it is desirable to keep the hydrodynamic sizeof the ligand low to facilitate extravazation from the blood stream.Alternatively, where it is desired to have the ligand remain in thesystemic circulation for a longer period of time the size of the ligandcan be increased, for example by formatting as an Ig like protein.

Half-life extension by targeting an antigen or epitope that increaseshalf-live in vivo: The hydrodynamic size of a ligand and its serumhalf-life can also be increased by conjugating or associating anTGFbetaRII binding polypeptide, dAb or ligand of the disclosure to abinding domain (e.g., antibody or antibody fragment) that binds anantigen or epitope that increases half-live in vivo, as describedherein. For example, the TGFbetaRII binding agent (e.g., polypeptide)can be conjugated or linked to an anti-serum albumin or anti-neonatal Fcreceptor antibody or antibody fragment, e.g. an anti-SA or anti-neonatalFc receptor dAb, Fab, Fab′ or scFv, or to an anti-SA affibody oranti-neonatal Fc receptor Affibody or an anti-SA avimer, or an anti-SAbinding domain which comprises a scaffold selected from, but not limitedto, the group consisting of CTLA-4, lipocallin, SpA, an affibody, anavimer, GroEI and fibronectin (see WO2008096158 for disclosure of thesebinding domains, which domains and their sequences are incorporatedherein by reference and form part of the disclosure of the presenttext). Conjugating refers to a composition comprising polypeptide, dAbor antagonist of the disclosure that is bonded (covalently ornoncovalently) to a binding domain such as a binding domain that bindsserum albumin.

Typically, a polypeptide that enhances serum half-life in vivo is apolypeptide which occurs naturally in vivo and which resists degradationor removal by endogenous mechanisms which remove unwanted material fromthe organism (e.g., human). For example, a polypeptide that enhancesserum half-life in vivo can be selected from proteins from theextracellular matrix, proteins found in blood, proteins found at theblood brain barrier or in neural tissue, proteins localized to thekidney, liver, lung, heart, skin or bone, stress proteins,disease-specific proteins, or proteins involved in Fc transport.Suitable polypeptides are described, for example, in WO2008/096158.

Such an approach can also be used for targeted delivery of a singlevariable domain, polypeptide or ligand in accordance with the disclosureto a tissue of interest. In one embodiment targeted delivery of a highaffinity single variable domain in accordance with the disclosure isprovided.

dAbs that Bind Serum Albumin: The disclosure in one embodiment providesa polypeptide or antagonist (e.g., dual specific ligand comprising ananti-TGFbetaRII dAb (a first dAb)) that binds to TGFbetaRII and a seconddAb that binds serum albumin (SA), the second dAb binding SA. Details ofdual specific ligands are found in WO03002609, WO04003019, WO2008096158and WO04058821.

In particular embodiments of the ligands and antagonists, the dAb bindshuman serum albumin and competes for binding to albumin with a dAbselected from the group consisting of any of the dAb sequences disclosedin WO2004003019 (which sequences and their nucleic acid counterpart areincorporated herein by reference and form part of the disclosure of thepresent text), any of the dAb sequences disclosed in WO2007080392 (whichsequences and their nucleic acid counterpart are incorporated herein byreference and form part of the disclosure of the present text), any ofthe dAb sequences disclosed in WO2008096158 (which sequences and theirnucleic acid counterpart are incorporated herein by reference and formpart of the disclosure of the present text).

In certain embodiments, the dAb binds human serum albumin and comprisesan amino acid sequence that has at least about 80%, or at least about85%, or at least about 90%, or at least about 95%, or at least about96%, or at least about 97%, or at least about 98%, or at least about 99%amino acid sequence identity with the amino acid sequence of a dAbdescribed in any of WO2004003019, WO2007080392 or WO2008096158. Forexample, the dAb that binds human serum albumin can comprise an aminoacid sequence that has at least about 90%, or at least about 95%, or atleast about 96%, or at least about 97%, or at least about 98%, or atleast about 99% amino acid sequence identity with the amino acidsequence of any of these dAbs. In certain embodiments, the dAb bindshuman serum albumin and comprises an amino acid sequence that has atleast about 80%, or at least about 85%, or at least about 90%, or atleast about 95%, or at least about 96%, or at least about 97%, or atleast about 98%, or at least about 99% amino acid sequence identity withthe amino acid sequence of the amino acid sequence of any of these dAbs.

In more particular embodiments, the dAb is a Vκ dAb that binds humanserum albumin. In more particular embodiments, the dAb is a V_(H) dAbthat binds human serum albumin.

Suitable Camelid V_(HH) that bind serum albumin include those disclosedin WO2004041862 (Ablynx N.V.) and in WO2007080392 (which V_(HH)sequences and their nucleic acid counterpart are incorporated herein byreference and form part of the disclosure of the present text). Incertain embodiments, the Camelid V_(HH) binds human serum albumin andcomprises an amino acid sequence that has at least about 80%, or atleast about 85%, or at least about 90%, or at least about 95%, or atleast about 96%, or at least about 97%, or at least about 98%, or atleast about 99% amino acid sequence identity with those sequencesdisclosed in WO2007080392 or any one of SEQ ID NOS:518-534, thesesequence numbers corresponding to those cited in WO2007080392 or WO2004041862.

In an alternative embodiment, the antagonist or ligand comprises abinding moiety specific for TGFbetaRII (e.g., human TGFbetaRII), whereinthe moiety comprises non-immunoglobulin sequences as described inWO2008096158, the disclosure of these binding moieties, their methods ofproduction and selection (e.g., from diverse libraries) and theirsequences are incorporated herein by reference as part of the disclosureof the present text).

Conjugation to a half-life extending moiety (e.g., albumin): In oneembodiment, a (one or more) half-life extending moiety (e.g., albumin,transferrin and fragments and analogues thereof) is conjugated orassociated with the TGFbetaRII-binding polypeptide, dAb or antagonist ofthe disclosure. Examples of suitable albumin, albumin fragments oralbumin variants for use in a TGFbetaRII-binding format are described inWO2005077042, which disclosure is incorporated herein by reference andforms part of the disclosure of the present text.

Further examples of suitable albumin, fragments and analogs for use in aTGFbetaRII-binding format are described in WO 03076567, which disclosureis incorporated herein by reference and which forms part of thedisclosure of the present text.

Where a (one or more) half-life extending moiety (e.g., albumin,transferrin and fragments and analogues thereof) is used to format theTGFbetaRII-binding polypeptides, dAbs and antagonists of the disclosure,it can be conjugated using any suitable method, such as, by directfusion to the TGFbetaRII-binding moiety (e.g., anti-TGFbetaRII dAb), forexample by using a single nucleotide construct that encodes a fusionprotein, wherein the fusion protein is encoded as a single polypeptidechain with the half-life extending moiety located N- or C-terminally tothe TGFbetaRII binding moiety. Alternatively, conjugation can beachieved by using a peptide linker between moieties, e.g., a peptidelinker as described in WO03076567 or WO2004003019 (these linkerdisclosures being incorporated by reference in the present disclosure toprovide examples for use in the present disclosure).

Conjugation to PEG: In other embodiments, the half-life extending moietyis a polyethylene glycol moiety. In one embodiment, the antagonistcomprises (optionally consists of) a single variable domain of thedisclosure linked to a polyethylene glycol moiety (optionally, whereinsaid moiety has a size of about 20 to about 50 kDa, optionally about 40kDa linear or branched PEG). Reference is made to WO04081026 for moredetail on PEGylation of dAbs and binding moieties. In one embodiment,the antagonist consists of a dAb monomer linked to a PEG, wherein thedAb monomer is a single variable domain according to the disclosure.

In another embodiment, a single variable domain, ligand or polypeptidein accordance with the disclosure may be linked to a toxin moiety ortoxin.

Protease resistance: Single variable domains, polypeptides or ligands inaccordance with the disclosure can be modified to improve theirresistance to protease degradation. As used herein, a peptide orpolypeptide (e.g. a domain antibody (dAb)) that is “resistant toprotease degradation” is not substantially degraded by a protease whenincubated with the protease under conditions suitable for proteaseactivity. A polypeptide (e.g., a dAb) is not substantially degraded whenno more than about 25%, no more than about 20%, no more than about 15%,no more than about 14%, no more than about 13%, no more than about 12%,no more than about 11%, no more than about 10%, no more than about 9%,no more than about 8%, no more than about 7%, no more than about 6%, nomore than about 5%, no more than about 4%, no more than about 3%, nomore that about 2%, no more than about 1%, or substantially none of theprotein is degraded by protease after incubation with the protease forabout one hour at a temperature suitable for protease activity. Forexample at 37 or 50 degrees C. Protein degradation can be assessed usingany suitable method, for example, by SDS-PAGE or by functional assay(e.g., ligand binding) as described herein.

Methods for generating dAbs with enhanced protease resistance aredisclosed, for example, in WO2008149143. In one embodiment, the singlevariable domain, polypeptide or ligand in accordance with the disclosureis resistant to degradation by leucozyme and/or trypsin. Polypeptides,immunoglobulin single variable domains and ligands of the disclosure maybe resistant to one or more of the following: serine protease, cysteineprotease, aspartate proteases, thiol proteases, matrix metalloprotease,carboxypeptidase (e.g., carboxypeptidase A, carboxypeptidase B),trypsin, chymotrypsin, pepsin, papain, elastase, leukozyme, pancreatin,thrombin, plasmin, cathepsins (e.g., cathepsin G), proteinase (e.g.,proteinase 1, proteinase 2, proteinase 3), thermolysin, chymosin,enteropeptidase, caspase (e.g., caspase 1, caspase 2, caspase 4, caspase5, caspase 9, caspase 12, caspase 13), calpain, ficain, clostripain,actimidain, bromelain, and separase. In particular embodiments, theprotease is trypsin, elastase or leucozyme. The protease can also beprovided by a biological extract, biological homogenate or biologicalpreparation. Polypeptides, immunoglobulin single variable domains andligands as disclosed herein may be selected in the presence of lungproteases, such that said polypeptides, immunoglobulin single variabledomains and ligands are resistant to said lung proteases. In oneembodiment, the protease is a protease found in sputum, mucus (e.g.,gastric mucus, nasal mucus, bronchial mucus), bronchoalveolar lavage,lung homogenate, lung extract, pancreatic extract, gastric fluid,saliva. In one embodiment, the protease is one found in the eye and/ortears. Examples of such proteases found in the eye include caspases,calpains, matrix metalloproteases, disintegrin, metalloproteinases (e.g.ADAMs—a disintegrin and metalloproteinase) and ADAM with thrombospondinmotifs, the proteosomes, tissue plasminogen activator, secretases,cathepsin B and D, cystatin C, serine protease PRSS1, ubiquitinproteosome pathway (UPP). In one embodiment, the protease is a nonbacterial protease. In an embodiment, the protease is an animal, e.g.,mammalian, e.g., human, protease. In an embodiment, the protease is a GItract protease or a pulmonary tissue protease, e.g., a GI tract proteaseor a pulmonary tissue protease found in humans. Such protease listedhere can also be used in the methods described, for example, inWO2008149143, involving exposure of a repertoire of library to aprotease.

Stability: In one aspect of the disclosure, the polypeptides, singlevariable domains, dAbs, ligands, compositions or formulations of thedisclosure are substantially stable after incubation (at a concentrationof polypeptide or variable domain of 1 mg/ml) at 37 to 50° C. for 14days in Britton Robinson or PBS buffer. In one embodiment, at least 65,70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%of the polypeptide, antagonist or variable domain etc. remainsunaggregated after such incubation at 37 degrees C. In one embodiment,at least 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99% of the polypeptide or variable domain remains monomericafter such incubation at 37 degrees C.

In one embodiment, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99% of the polypeptide, antagonist or variable domain remainsunaggregated after such incubation at 50 degrees C. In one embodiment,at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of thepolypeptide or variable domain remains monomeric after such incubationat 50 degrees C. In one embodiment, no aggregation of the polypeptides,variable domains, antagonists is seen after any one of such incubations.In one embodiment, the pI of the polypeptide or variable domain remainsunchanged or substantially unchanged after incubation at 37 degrees C.at a concentration of polypeptide or variable domain of 1 mg/ml inBritton-Robinson buffer. In one aspect of the disclosure, thepolypeptides, variable domains, antagonists, compositions orformulations of the disclosure are substantially stable after incubation(at a concentration of polypeptide or variable domain of 100 mg/ml) at4° C. for 7 days in Britton Robinson buffer or PBS at a pH of 7 to 7.5(e.g., at pH7 or pH7.5). In one embodiment, at least 95, 95.5, 96, 96.5,97, 97.5, 98, 98.5, 99 or 99.5% of the polypeptide, antagonist orvariable domain remains unaggregated after such incubation. In oneembodiment, at least 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99 or 99.5%of the polypeptide or variable domain remains monomeric after suchincubation. In one embodiment, no aggregation of the polypeptides,variable domains, antagonists is seen after any one of such incubations.

In one aspect of the disclosure, the polypeptides, variable domains,antagonists, compositions or formulations of the disclosure aresubstantially stable after nebulisation (e.g. at a concentration ofpolypeptide or variable domain of 40 mg/ml) e.g., at room temperature,20 degrees C. or 37° C., for 1 hour, e.g. jet nebuliser, e.g. in a PariLC+ cup. In one embodiment, at least 65, 70, 75, 80, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99 or 99.5%of the polypeptide, antagonist or variable domain remains unaggregatedafter such nebulisation. In one embodiment, at least 65, 70, 75, 80, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5, 97, 97.5, 98,98.5, 99 or 99.5% of the polypeptide or variable domain remainsmonomeric after such nebulisation. In one embodiment, no aggregation ofthe polypeptides, variable domains, antagonists is seen after any one ofsuch nebulisation.

Monomeric form: In one embodiment, the dAb of the present disclosure isidentified to be preferentially monomeric. Suitably, the disclosureprovides a (substantially) pure monomer. In one embodiment, the dAb isat least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% pure or 100% puremonomer. To determine whether dAbs are monomeric or form higher orderoligomers in solution, they can be analysed by SEC-MALLS. SEC MALLS(size exclusion chromatography with multi-angle-LASER-light-scattering)is a non-invasive technique for the characterizing of macromolecules insolution, that is familiar to any skilled in the art. Briefly, proteins(at concentration of 1 mg/mL in buffer Dulbecco's PBS) are separatedaccording to their hydrodynamic properties by size exclusionchromatography (column: TSK3000; S200). Following separation, thepropensity of the protein to scatter light is measured using amulti-angle-LASER-light-scattering (MALLS) detector. The intensity ofthe scattered light while protein passes through the detector ismeasured as a function of angle. This measurement taken together withthe protein concentration determined using the refractive index (RI)detector allows calculation of the molar mass using appropriateequations (integral part of the analysis software Astra v.5.3.4.12).

Therapeutic use: The disclosure provides a method for treating,suppressing or preventing diseases associated with TGFbeta signaling. Inone embodiment, such disease may be caused or contributed to bydysregulated TGFbeta signaling, by overexpression of TGFbeta or by highlevels of bioavailable TGFbeta. Diseases associated with TGFbetasignaling include diseases relating to fibroses of various tissues, suchas pulmonary fibrosis including idiopathic pulmonary fibrosis (IPF) andother interstitial lung disease such as acute respiratory distresssyndrome (ARDS), fibrosis of the liver including cirrhosis and chronichepatitis, rheumatoid arthritis, ocular disorders, vascular conditionssuch as restenosis, fibrosis of the skin including keloid of skin andscarring following wound healing and Dupuytren's Contracture, and kidneysuch as nephritis, kidney fibrosis and nephrosclerosis or a vascularcondition such as restenosis. Other diseases associated with TGFbetasignaling include vascular diseases such as hypertension, pre-eclampsia,hereditary haemorrhagic telangtiectasia type I (HHT1), HHT2, pulmonaryarterial hypertension, aortic aneurysms, Marfan syndrome, familialaneurysm disorder, Loeys-Dietz syndrome, arterial tortuosity syndrome(ATS). Other diseases associated with TGFbeta signaling include diseasesof the musculoskeletal system such as Duchenne's muscular dystrophy andmuscle fibrosis. Further diseases associated with TGFbeta signalinginclude cancer such as colon, gastric and pancreatic cancer as well asglioma and NSCLC. In addition, the disclosure provides methods fortargeting cancer by, for example, modulating TGFbeta signaling in thetumour angiogenesis or through treatment of the cancer stroma. Otherdiseases or conditions include those related to tissue scarring. Otherdiseases include pulmonary diseases such as COPD (Chronic obstructivepulmonary disease), liver diseases such as liver failure (e.g. viralhepatitis, alcohol, obesity, autoimmune, metabolic, obstructive), kidneydiseases including renal failure (e.g. diabetes, hypertension),hypertrophic cardiomyopathy, transplant rejection (lung/liver/kidney)and hypertrophic and keloid scarring.

“Fibrosis” is the result of excess deposition of extracellular matrixcomponents such as collagen causing overgrowth, scarring and/orhardening of tissues.

“Skin Fibrosis”: cutaneous fibrosis covers a variety of human disorderswith differing aetiology, but with a common dysregulation of connectivetissue metabolism, particularly of dermal fibroblasts. Specific examplesof cutaneous fibrosis include keloid disease, hypertrophic scars (HS)and scleroderma. Keloid disease and hypertrophic scars, although notsubgroups of the same condition are both resultant from scarringfollowing wound healing, with Keloids spreading beyond the originalwound site whilst hypertrophic scar is constrained within the margins ofthe original wound. Scleroderma, however is used to describe fibrosis ofthe skin in systemic sclerosis which is a systemic condition resultingin fibrosis of multiple organs. In an embodiment, the variable domain,ligand, fusion protein or polypeptide as disclosed herein is used toprevent or treat keloid disease, hypertrophic scars or scleroderma.

“Keloids” are fibrous overgrowths at sites of cutaneous injury that formas a result of an abnormal wound-healing process in geneticallysusceptible individuals and, unlike normal scars, do not regress.Predominantly observed in patients with darkly pigmented skin, “Keloiddisease” is a benign dermal fibroproliferative tumor unique to humansthat never becomes malignant.

“Dupuytren's contracture” is a localized formation of scar tissuebeneath the skin of the palm of the hand. The scarring accumulates in atissue (fascia) that normally covers the tendons that pull the fingersto grip. As Dupuytren contracture progresses, more of the fascia becomesthickened and shortened, resulting in flexion contracture of the handwhere the fingers bend towards the palm and cannot be fully extended(straightened), resulting in extreme cases to loss of use of the hand.

Scarring occurs following, surgery, injury or trauma to tissues ororgans within the body. They are a consequence of repair mechanisms thatgenerate extracellular matrix to replace missing normal tissue. The skinrepresents the most frequently injured tissue resulting in dermalscarring, which can result in adverse consequences including: loss offunction; contracture; and, poor aesthetics which may have causepsychological effects to the sufferer. Scars can be defined ‘amacroscopic disturbance of the normal structure and function of the skinarchitecture, resulting from the end product of wound healing’(Fergusson et al., 1996). Currently no therapies exist to prevent orimprove scarring effectively.

The role of TGFbeta in pulmonary fibrosis has been observed (Wynn etal., J. Pathology 2008, 214, p. 199-210; Sime et al. J. ClinicalImmunology 1997, Vol. 100, p. 768-776). A shift to increased productionof Th2 cytokines and decreased production of Th1 cytokines is observedas a result of unknown lung injury. Overexpression of TGFbeta stimulatesangiogenesis, fibroblast activation, deposition of ECM, andfibrogenesis. Animal models (e.g. TGFbeta overexpression, SMAD3 KO,inhibition of TGFbetaR signaling) show that TGFbeta is a key mediatorfor the development of pulmonary fibrosis.

“Idiopathic pulmonary fibrosis (IPF)” is a chronic and progressivedisease resulting in abnormal and excessive deposition of fibrotictissue in the pulmonary interstitium without a known cause. There is anincidence of approximately 10-20 cases per 100,000 in U.S per year. Theprevalence increases sharply with age, reaching 175 cases per 100,000over the age of 75 with onset usually occurring between 50 and 70 yrs.The five year survival rate is 20% with a mean survival of 2.8 years.Symptoms include a dry cough and progressive breathlessness, abnormalchest x-ray or HRCT and reduced lung volumes. Current treatments includecorticosteroids (Prednisone), immunosuppressives (cyclophosphamide) ortransplantation although none of the currently available therapies havea proven efficacy. In one embodiment, the single variable domain orpolypeptide of the present disclosure provides a treatment for IPF.

Suitably, a successful treatment for Idiopathic pulmonary fibrosis (IPF)will show any one of a decrease in lung fibroblast proliferation, anincrease in lung fibroblast apoptosis, a decrease in excessiveextracellular matrix synthesis and deposition, an increase inextracellular matrix breakdown and remodelling or will show someprotection against ongoing tissue injury and restoration of normalhistopathology.

Suitably, a successful treatment would decelerate disease progression.

The efficacy of a treatment for IPF can be demonstrated in the bleomycininduced pulmonary fibrosis model. In one embodiment, the immunoglobulinsingle variable domain of the present disclosure cross reacts with mouseTGFbetaRII such that its efficacy can be tested in the mouse model.

TGFbeta is an important cell signaling molecule in the modulation ofcell behaviour in ocular tissues. Overactivation of TGFbeta isimplicated in the pathogenesis of fibrotic diseases in eye tissue whichcan be wound healing-related and lead to impaired vision and oculartissue homeostasis (reviewed, for example, by Saika, LaboratoryInvestigation (2006), 86, 106-115).

Accordingly, in one embodiment, diseases associated with TGFbetasignaling include ocular disorders such as fibrotic diseases of the eyetissue. Fibrotic disease of the eye may occur in the cornea,conjunctiva, lens or retina. Ocular disorders include proliferativevitreoretinopathy (PVR), a disorder of post-retinal detachment andretinal fibrosis, diabetic retinopathy, glaucoma, such as open-angleglaucoma, angle-closure, congenital and pseudo-exfoliation syndrome,wound healing reactions in the lens, such as post chemical or thermalburn, or Stevens-Johnson's syndrome, and post-cataract surgerycomplications. TGFbeta also has a role in cataract development(Wormstone et al. Exp Eye Res; 83 1238-1245, 2006). A number of oculardisorders occur as a result of fibrosis post surgery. In addition, overactivity of TGFbeta2 (transforming growth factor β2) is believed tocause scarring in and around the eye after glaucoma filtration surgery.TGFbeta2 is the predominant isoform involved in pathological scarring ofocular tissues including the cornea, retina, conjunctiva and trabelularmeshwork. Scarring or fibrosis of the trabelular meshwork can lead toocclusion of the normal aqueous outflow pathway leading to raisedintraocular pressure and risk of glaucoma development. TGFbeta 2 hasbeen shown to be a pathological agent in pre-clinical models of glaucomadisease. TGFbeta2 levels are elevated in patients with glaucoma, invitro treatment of huTM cells with TGFbeta-2 leads to phenotypic changesand upregulation of ECM modulating proteins (MMP-2, PAI-I)(Lutjen-Drecol (2005), Experimental Eye Research, Vol. 81, Issue 1,pages 1-4; Liton (2005), Biochemical and Biophysical ResearchCommunications Vol. 337, issue 4, p. 1229-1236; Fuchshofer et al (2003),Experimental Eye Research, Vol. 77, issue 6, p. 757-765; Association forResearch in Vision and Ophthalmology (ARVO) conference poster #16312009). Moreover, overexpression of TGFbeta in the eye leads toglaucoma-like pathology in mice (ARVO conference poster #5108 2009) anddelivery of TGFbeta-2 using AAV has been shown to inhibit retinalganglion cell loss in a rat model of glaucoma (ARVO conference poster#5510 2009). More recently, oxidative stress induction in cultured humanoptic nerve head astrocytes has been shown to increase TFGbeta2secretion (Yu et al (2009) Invest. Ophthalmol. Vis. Sci. 50: 1707-1717).This all indicates that reduction of TGFbeta 2 levels might minimize thecharacteristic optic nerve head changes seen in glaucoma. However,TGFbeta is also known to have an immunosuppressive role and so in someaspects can be protective so a reduction in elevated levels of TGFbeta2rather than a complete knock down may be preferred in treatment ofchronic ocular conditions such as glaucoma. Accordingly, diseases whichcan be treated using the dAbs and compositions etc. in accordance withthe disclosure include scarring post glaucoma filtration surgery.

Accordingly, in one aspect there is provided a method for treating,suppressing or preventing a disease associated with TGFbeta signalingand, in particular, dysregulated TGFbeta signaling, comprisingadministering to a mammal in need thereof a therapeutically-effectivedose or amount of a polypeptide, fusion protein, single variable domain,antagonist or composition according to the disclosure.

In another aspect, the disclosure provides an immunoglobulin singlevariable domain, polypeptide, ligand or fusion protein in accordancewith the disclosure for use as a medicament. Suitable a medicament maycomprise an immunoglobulin single variable domain etc. in accordancewith the disclosure formatted as described herein.

Suitably, the medicament is a pharmaceutical composition. In a furtheraspect of the disclosure, there is provided a composition (e.g.,pharmaceutical composition) comprising a polypeptide, single variabledomain, ligand, composition or antagonist according to the disclosureand a physiologically or pharmaceutically acceptable carrier, diluent orexcipient. In one embodiment, the composition comprises a vehicle fordelivery. In particular embodiments, the polypeptide, fusion protein,single variable domain, antagonist or composition is administered viapulmonary delivery, such as by inhalation (e.g., intrabronchial,intranasal or oral inhalation, intranasal such as by drops) or bysystemic delivery (e.g., parenteral, intravenous, intramuscular,intraperitoneal, intraarterial, intrathecal, intraarticular,subcutaneous, vaginal or rectal administration). In another embodiment,the polypeptide, single variable domain, ligand or fusion protein orcompositions in accordance with the disclosure is administered to theeye e.g. by topical administration, as eye drops, particulate polymersystem, gel or implant, or by intraocular injection e.g. into thevitreous humour. Delivery can be targeted to particular regions of theeye such as the surface of the eye, or the tear ducts or lacrimal glandsor to the anterior or posterior chambers of the eye such as the vitreoushumour). It can also be useful if the immunoglobulin single variabledomain, composition etc. is delivered to the eye along with an ocularpenetration enhancer e.g. sodium caprate or with a viscosity enhancere.g. Hydroxypropylmethylcellulose (HPMC). In further embodiments, thepolypeptide, fusion protein, single variable domain, antagonist orcomposition is administered to the skin; by topical delivery to thesurface of the skin and/or delivery to a region(s) within the skin e.g.intradermal delivery.

Although the most accessible organ of the body for delivery, the skin'soutermost barrier, the stratum corneum (SC), acts as a rate limitingbarrier for drug delivery. Traditionally, intradermal injection has beenrequired to circumvent the SC allowing delivery of drug to site ofaction in deeper skin layers. Delivery however, maybe achieved throughother transdermal delivery approaches. Formulation methodologies maybeutilised for delivery, including: chemical enhancers to alter the lipidstructure of the SC; peptide facilitators enabling transfolliculartransport; and encapsidation in particles including, liposome's,niosomes, ethosomes and transfersomes, which are believed to aid localfluidisation of the lipids and formation of depots for prolonged effect.Iontophoresis, involving the application of a small electrical potentialacross the skin, has been used for localised drug delivery.Iontophoresis allows for both the delivery of charged and neutralmolecules by electromigration and electroosmosis respectively.Microneedles, can be employed to create micron-sized channels in theskin to overcome the SC, allowing proteins to pass through thesechannels to the lower epidermis. Microneedles can be broadly classifiedinto solid and hollow microneedles. Solid microneedles, maybe used todisrupt the SC, prior to drug administration, coated to allow deliveryas drug dissolves from the needles, or soluble allowing drug release asthe needles dissolve in situ. Hollow microneedles allow for infusion ofa liquid formulation of drug substance. Electroporation, unlikeiontophoresis requires higher voltages >50V, to alter skin permeabilityin order to enhance drug penetration. Thermal and radiofrequencyablation methodologies allow for disruption of the SC through localisedheating and ablation of the SC. In heat ablation this results followingapplication of high temperature for short periods of time, whereasradiofrequency ablation involves use of radiofrquencies, to vibratemicroelectrodes on the skin, resulting in localised heating. Disruptionof the SC can also be achieved through Laser abrasion, application oflow frequency ultrasound waves (sonophoresis) and jet injectorsutilising high velocities to propel drug through the SC.

Moreover, the present disclosure provides a method for the treatment ofdisease using a polypeptide, single variable domain, composition, ligandor antagonist according to the present disclosure. In one embodiment thedisease is a tissue fibrosis such as keloid disease or Dupuytren'sContracture.

In an aspect of the disclosure, the polypeptide, single variable domain,ligand, composition or antagonist is provided for therapy and/orprophylaxis of a disease or condition associated with TGFbeta signalingin a human. In another aspect, there is provided the use of thepolypeptide, single variable domain, composition or antagonist, in themanufacture of a medicament for therapy or prophylaxis of a disease orcondition associated with TGFbeta signaling in a human. In anotheraspect, there is provided a method of treating and/or preventing adisease or condition associated with TGFbeta signaling in a humanpatient, the method comprising administering the polypeptide, singlevariable domain, composition or antagonist to the patient. Thedisclosure also relates to therapeutic methods that compriseadministering a therapeutically effective amount of a ligand of thedisclosure (e.g., antagonist, or single variable domain) to a subject inneed thereof.

In other embodiments, the disclosure relates to a method for treatingidiopathic pulmonary fibrosis comprising administering to a subject inneed thereof a therapeutically effective amount of a ligand of thedisclosure (e.g., antagonist, or single variable domain).

The disclosure also relates to a drug delivery device comprising thecomposition (e.g., pharmaceutical composition) of the disclosure. Insome embodiments, the drug delivery device comprises a plurality oftherapeutically effective doses of ligand.

In other embodiments, the drug delivery device is selected from thegroup consisting of parenteral delivery device, intravenous deliverydevice, intramuscular delivery device, intraperitoneal delivery device,transdermal or intradermal delivery device, pulmonary delivery device,intraarterial delivery device, intrathecal delivery device,intraarticular delivery device, subcutaneous delivery device, intranasaldelivery device, ocular delivery device, vaginal delivery device, rectaldelivery device, syringe, a transdermal delivery device, an intradermaldelivery device, a capsule, a tablet, a nebulizer, an inhaler, anatomizer, an aerosolizer, a mister, a dry powder inhaler, a metered doseinhaler, a metered dose sprayer, a metered dose mister, a metered doseatomizer, and a catheter. In an embodiment the drug delivery device is atransdermal or intradermal delivery device.

Suitably, the disclosure provides a pulmonary delivery device containinga polypeptide, single variable domain, composition or antagonistaccording to the disclosure. The device can be an inhaler or anintranasal administration device. Suitably, the pulmonary deliverydevice enables delivery of a therapeutically effective dose of a ligandetc. in accordance with the disclosure.

In another embodiment, the disclosure provides an ocular delivery devicecontaining a polypeptide, single variable domain, composition orantagonist according to the disclosure. Suitably, the ocular deliverydevice enables delivery of a therapeutically effective dose of a ligandetc. in accordance with the disclosure.

As used herein, the term “dose” refers to the quantity of ligandadministered to a subject all at one time (unit dose), or in two or moreadministrations over a defined time interval. For example, dose canrefer to the quantity of ligand (e.g., ligand comprising animmunoglobulin single variable domain that binds TGFbetaRII)administered to a subject over the course of one day (24 hours) (dailydose), two days, one week, two weeks, three weeks or one or more months(e.g., by a single administration, or by two or more administrations).The interval between doses can be any desired amount of time. In aparticular embodiment, the single variable domain or polypeptide of theinvention is administered into the skin by injection, in particular byintradermal delivery, weekly or fortnightly or every 7-10 days, forexample every 7, 8, 9 or 10 days.

In one embodiment, the single variable domain of the disclosure isprovided as a dAb monomer, optionally unformatted (e.g., not PEGylatedor half-life extended) or linked to a PEG, optionally as a dry powderformulation, optionally for delivery to a patient by inhalation (e.g.,pulmonary delivery), optionally for treating and/or preventing a lungcondition (e.g., Idiopathic pulmonary fibrosis).

The ligands of the disclosure provide several advantages. For example,as described herein, the ligand can be tailored to have a desired invivo serum half-life. Domain antibodies are much smaller thanconventional antibodies, and can be administered to achieve bettertissue penetration than conventional antibodies. Thus, dAbs and ligandsthat comprise a dAb provide advantages over conventional antibodies whenadministered to treat disease, such as TGFbeta-signaling-mediateddisease. In particular, pulmonary delivery of a dAb of the presentdisclosure to treat idiopathic pulmonary fibrosis enables specific localdelivery of an inhibitor of TGFbeta signaling. Advantageously, anunformatted dAb monomer which specifically binds to and inhibitsTGFbetaRII is small enough to be absorbed into the lung throughpulmonary delivery.

The examples of WO2007085815 are incorporated herein by reference toprovide details of relevant assays, formatting and experiments that canbe equally applied to ligands of the present disclosure.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though fully set forth.

The disclosure is further described, for the purposes of illustrationonly, in the following examples.

EXAMPLES Example 1 Selection of dAbs which Bind TGFbetaRII

Selection of dAbs which Bind Mouse TGFbetaRII

Naïve Selections: 4G and 6G naïve phage libraries, phage librariesdisplaying antibody single variable domains expressed from the GAS1leader sequence (see WO2005093074) for 4G and additionally withheat/cool preselection for 6G (see WO04101790), were used. The DOM23leads were isolated by panning pools of VH and VK libraries (identifiedas 4G H11-19 and 6G VH2-4 (VH dAbs) and 4G κ1, 4G κ2 and 6G κ (Vκ dAbs)against the recombinant mouse and human TGF-β RII/Fc chimera protein.These chimeric proteins were made by expression of a DNA sequenceencoding the amino acids residues 24 to 159 of the extracellular domainof human TGF-β Receptor Type II (Lin, et al, 1992, Cell 68:775-785)fused to the Fc region of human IgG1 in a human embryonic kidney cellline, HEK-F.

The recombinant mouse and human TGF-β RII/Fc chimera proteins werebiotinylated using EZ-LINK™ Sulfo-NHS-LC-Biotin reagent (Pierce,Rockford, USA) (Henderikx, et al., 2002, Selection of antibodies againstbiotinylated antigens. Antibody Phage Display: Methods and protocols,Ed. O'Brien and Atkin, Humana Press). The phage libraries were pooledinto six groups; 4G κ1 and κ2, 6G κ, 4G H11-13, 4G H14-16, 4G H17-19 and6G VH2-4. 1×10¹¹ phage per library were pooled.

The phage were blocked in 2% MARVEL™ milk powder in phosphate bufferedsaline (MPBS) with the addition of 10 uM Human IgG Fc fragment (NativeIgG Fc fragment derived from human myeloma plasma IgG, Calbiochem,California, US, cat. no. 401104) for one hour. 200 nM biotinylated mouseTGF-β RII/Fc was incubated with the blocked phage and Fc fragmentmixture for 1 hour at room temperature and then captured on streptavidinDYNAbeads™ (Dynal, UK) for five minutes. The beads were washed seventimes with 1 ml phosphate buffered saline/0.1% TWEEN™ (PBST), followedby a wash with 1 ml phosphate buffered saline (PBS). The biotinylatedmouse TGF-β RII/Fc-bound phage were eluted in 500 ul 1 mg/ml trypsin inPBS for 10 minutes and then used to infect 1.75 ml of log-phaseEscherichia coli TG1 for 30 minutes. Cells were plated on 2×TYE (TryptonYeast Extract) agar plates supplemented with 15 ug/ml tetracycline. Forsubsequent rounds of selections, cells were scraped from the plates andused to inoculate 50 ml 2×TY (Trypton Yeast)+15 ug/ml tetracyclinecultures that were grown overnight at 37° C. for phage amplification.

Amplified phage was recovered by centrifugation of the overnight culturefor 10 minutes at 4566 g. 40 ml of supernatant containing the amplifiedphage was added to 10 ml of PEG/NaCl (20% v/w PEG 8000+2.5M NaCl) andincubated on ice for 45 to 60 minutes. The samples were centrifuged for30 minutes at 4566 g to pellet the precipitated phage. The supernatantwas discarded and the phage pellet was resuspended in 2 ml 15% v/vglycerol/PBS. The phage sample was transferred to 2 ml Eppendorf tubesand centrifuged for 10 minutes at g to remove any remaining bacterialcell debris. The phage was used as input phage for the second round ofselection. The second round of selection was performed as described forthe first round, except approximately 1×10¹⁰ phage were added, andeither 200 nM human TGF-β RII/Fc or 20 nM mouse TGF-β RII/Fc was used inthe selections.

Second round outputs were cloned from the fd-phage vector, pDOM4 intopDOM10. Vector pDOM4, is a derivative of the fd phage vector in whichthe gene III signal peptide sequence is replaced with the yeastglycolipid anchored surface protein (GAS) signal peptide. It alsocontains a c-myc tag between the leader sequence and gene III, whichputs the gene III back in frame. This leader sequence functions wellboth in phage display vectors but also in other prokaryotic expressionvectors and can be universally used. pDOM10 is a plasmid vector designedfor soluble expression of dAbs. It is based on pUC119 vector, withexpression under the control of the LacZ promoter. Expression of dAbsinto the supernatant was ensured by fusion of the dAb gene to theuniversal GAS leader signal peptide (see WO2005093074) at the N-terminalend. In addition, a FLAG-tag was appended at the C-terminal end of thedAbs.

Subcloning of the dAb genes was performed by isolating pDOM4 DNA fromthe cells infected by the selected dAb-displaying fd-phage using aQIAPREP™ Spin MINIPREP™ kit in accordance with the manufacturer'sinstructions (cat. no. 27104, Qiagen). The DNA was amplified by PCRusing biotinylated oligonucleotides DOM57 (5′ TTGCAGGCGTGGCAACAGCG-3′(SEQ ID NO:197) and DOME (5′-CACGACGTTGTAAAACGACGGCC-3′ (SEQ IDNO:198)), digested with SalI and NotI restriction endonucleases andligated with pDOM10 digested with SalI and NotI. The ligation productswere transformed by electroporation into E coli HB2151 cells and platedon TYE plates (Trypton Yeast Extract) supplemented with 100 μg/ml ofcarbenicillin (TYE-carb). Individual clones were picked and expressed inovernight express auto-induction medium (high-level protein expressionsystem, Novagen), supplemented with 100 μg/ml carbenicillin. in 96-wellplates, grown with shaking at either 30° C. or 37° C. These expressionplates were then centrifuged at 1800 g for 10 minutes. dAb clones thatbound mouse and/or human TGF-β RII/Fc were identified by an ELISA andBIACORE™ (GE HEALTHCARE™) screen or by MSD (Meso Scale Discovery)binding assay screen. For the ELISA, 96-well Maxisorp™ immuno plates(Nunc, Denmark) were coated with either human or mouse TGF-β RII/Fcovernight at 4° C. The wells were washed three times with PBST and thenblocked with 1% TWEEN™ in PBS (1% TPBS) for 1 hour at room temperature.The block was removed and a 1:1 mixture of 1% TPBS and dAb supernatantwas added for 1 hour at room temperature. The plate was washed threetimes with PBST and the detection antibody (Monoclonal anti-FLAGM2-peroxidase antibody, Sigma-Aldrich, UK) was added and incubated for 1hour at room temperature. The plates were developed using acolourimetric substrate (SUREBLUE™ 1-component TMB Microwell Peroxidasesolution, KPL, Maryland, USA) and the optical density (OD) measured at450 nM, the OD₄₅₀ being proportional to the amount of bound detectionantibody. For BIACORE™, supernatants were diluted 1:1 in HBS-EP bufferand screened on BIACORE™ for binding to biotinylated human and mouseTGF-β RII/Fc (SA chip coated with 1500 Ru biotinylated hRII-Fc and 1550Ru biotinylated mRII-Fc in accordance with the manufacturer'srecommendations) (BIACORE™, GE HEALTHCARE™). Samples were run onBIACORE™ at a flow rate of 50 μl/min.

Naïve Human Selections and Screening

Selection of dAbs which Bind Human TGFbetaRII

Naïve selections were performed as described for mouse TGFbRII but using150 and 15 nM biotinylated human TGFbRII/Fc at round one and two,respectively. A third round was performed using the same method as forround two, but with 1.5 nM biotinylated human TGFbRII/Fc.

The third round outputs were cloned from the fd-phage vector, pDOM4 intopDOM10. Subcloning of the dAb genes was performed by isolating pDOM4 DNAfrom the cells infected by the selected dAb-displaying fd-phage using aQIAPREP™ Spin MIDIPREP™ kit in accordance with the manufacturer'sinstructions (cat. no. 27104, Qiagen). The plasmid DNA was digested withSalI and NotI restriction endonucleases and the dAb gene insert ligatedwith pDOM10 digested with SalI, NotI and PstI restriction endonucleases.The ligation products were transformed by electroporation into E. coliHB2151 cells and plated on TYE plates (Trypton Yeast Extract)supplemented with 100 μg/ml of carbenicillin (TYE-carb). Individualclones were picked and expressed in 96-well plates at 250 rpm, 30° C. 72hours, in 1 ml/well overnight express auto-induction medium (Novagen)supplemented with 100 μg/ml carbenicillin. These plates were thencentrifuged at 1800 g for 10 minutes. The soluble dAb supernatants werescreened for antigen binding in the TGFbRII MSD binding assay combinedwith the fluorescent polarization concentration determination assay. Thenumber of human TGFbRII binders was high and there were too many clonesto take forward for further characterization. Therefore, a subset ofclones was sequenced and those with unique sequences were furthercharacterized.

TGFβRII MSD Binding Assay

This assay was used to determine the binding activity of anti-TGFbRIIdAbs. TGFbRII-Fc antigen was coated onto a MSD plate, which wassubsequently blocked to prevent non-specific binding. Serially dilutedsupernatants containing soluble FLAG-tagged dAb were added. Afterincubation, the plate was washed and only dAbs that bound specificallyto TGFBRII-Fc remained bound to the plate. Bound dAbs were detected witha ruthenylated anti-FLAG tagged antibody and MSD read buffer. If theconcentration of the dAbs in the supernatant dilutions was determinedusing the Fluorescent Polarisation Concentration Determination assay,then concentration binding curves were plotted.

0.5 ul per well of either 60 μg/ml human TGFbRII-Fc, 60 μg/ml mouseTGFbRII-Fc or 60 ug/ml human IgG1 Fc (R&D systems, catalogue number110-HG) was spotted onto 384 well MSD high bind plates (Meso ScaleDiscovery). The plates were air-dried at room temperature for a minimumof four hours and no longer than overnight. The plates were blocked with50 ul per well of 5% MARVEL™ in Tris buffered saline (TBS)+0.1% TWEEN™20 for either 1 hour at room temperature or overnight at 4° C. Theblocking reagent was removed from the wells by flicking the plates. A1:3 dilution series of the dAb supernatants was prepared in 2×TY medium.The dAbs were expressed in the pDOM10 expression vector so were the dAbprotein was expressed as a FLAG fusion protein. The blocking reagent wasremoved and 10 ul per well of the diluted dAb supernatants weretransferred to the blocked MSD plates. The dAbs supernatants werescreened as either 4 point curves or as 11 point curves. In addition tothe diluted dAb supernatants, two controls were included in each plate,one low control (normalised to 0% binding), with no TGFbRII bindingspecificity and a high control (normalised to 100% binding) with highTGFbRII binding specificity, data not shown.

The plates were incubated with the dAb supernatants and the controlsamples for one hour at room temperature and then washed three timeswith 50 ul per well of TBS+0.1% TWEEN™. 15 ul/well of ruthenylatedanti-FLAG antibody was added to the plates and incubated for one hour atroom temperature. The anti-FLAG antibody (anti-FLAG M2 monoclonalantibody, Sigma, UK, catalogue number F3165) was conjugated to rutheniumII tris-bipyridine N-hydroxy succinimide following the manufacturer'sinstructions (Meso Scale Discovery, catalogue number R91BN-1). Theruthenylated anti-FLAG antibody was added to all wells except to themouse anti-human IgG1 Fc antibody control wells. Instead, 15 ul/well ofanti-Mouse MSD tag (Meso Scale Discovery, catalogue number R31AC-1) wereadded. The anti-Mouse MSD tag was diluted in 2% MARVEL™ in TBS+0.1%TWEEN™ 20 to a final concentration of 750 ng/ml. The plates wereincubated at room temperature for one hour and washed three times with50 ul per well of TBS+0.1% TWEEN™. 35 ul 1×MSD read buffer (Meso ScaleDiscovery) was added to each well and the plates were read on a MSDSector 6000 reader (Meso Scale Discovery).

Data were analysed using XC50 Activity Base. All data was normalised tothe mean of the high and low control wells on each plate, with the lowcontrol normalised to 0% binding and the high control normalised to 100%binding. A four parameter curve fit was applied to the normalised dataand concentration binding curves using dAb concentrations calculatedusing the Fluorescent Polarisation Concentration Determination of dAbsin supernatants assay, were plotted.

The four parameter fit used was as follows:

${y = {\frac{( {a - d} )}{1 + ( \frac{x}{c} )^{b}} + d}},$where a is the minimum, b is the Hill slope, c is the XC50 and d is themaximum.Fluorescence Polarisation Concentration Determination of dAbs inSupernatants Assay

This assay allows the concentration of soluble FLAG-tagged dAbsexpressed in supernatants to be determined. A fluorescentlylabelled-FLAG peptide was mixed with an anti-FLAG antibody. Thefluorescent molecules were excited with polarised light at a wavelengthof 531 nM and the emitted polarised light was read at a wavelength of595 nM. The addition of a FLAG-tagged dAb resulted in the displacementof the fluorescent peptide from the anti-FLAG antibody which in turnresulted in reduced polarisation of the emission signal. A standardcurve of known concentrations of purified FLAG-tagged VH dummy dAb wasprepared and was used to back calculate the concentration of the solubledAbs in the supernatants. The concentration data was combined withbinding activity data, allowing concentration binding curves to beplotted for dAb supernatants.

The dAb supernatants were serially diluted 1:2 in 2×TY medium (1:2, 1:4,1:8 and 1:16), followed by a 1:10 dilution in phosphate buffered saline(PBS). The diluted supernatants were transferred to a black 384 wellplate. A standard curve was set up by serially diluting purified VHDummy dAb 1:1.7 in 10% v/v 2×TY medium in PBS. The highest dAbconcentration was 10 uM and there were 16 dilutions in total. 5 ul ofeach dilution was transferred to the 384 well plate. A mixture of 5 nMFLAG peptide labelled at the c-terminus with Cy3b, 100 mM anti-FLAG M2monoclonal antibody (Sigma, catalogue number F3165), 0.4 mg/ml bovineserum albumin (BSA) in 2 mM CHAPs buffer was prepared. 5 ul of themixture was transferred to the wells containing the diluted dAbs (bothsupernatants and standard curve wells). The plate was centrifuged at1000 rpm (216 g) for 1 minute and then incubated in the dark at roomtemperature for 15 minutes. The plates were read on an ENVISION™ reader(Perkin Elmer) fitted with the following filters;

-   Excitation filter: BODIPY TMR FP 531-   Emission filter 1: BODIPY TMR FP P pol 595-   Emission filter 2: BODIPY TMR FP P pol 595-   Mirror: BODIPY TMR FP Dual Enh

The standard curve was plotted and used to back calculate theconcentrations of the soluble dAbs in the supernatants.

Mouse and human TGF-β RII/Fc-binding dAbs identified in the ELISA,BIACORE™ and MSD binding assays were expressed in overnight expressautoinduction medium (ONEX™, Novagen) at either 30° C. for 48 to 72hours. The cultures were centrifuged (4,600 rpm for 30 minutes) and thesupernatants were incubated with STREAMLINE™-protein A beads (AmershamBiosciences, GE HEALTHCARE™, UK. Binding capacity: 5 mg of dAb per ml ofbeads), either overnight at 4° C. or at room temperature for at leastone hour. The beads were packed into a chromatography column and washedwith either 1× or 2×PBS, followed by 10 or 100 mM Tris-HCl pH 7.4(Sigma, UK). Bound dAbs were eluted with 0.1 M glycine-HCl pH 2.0 andneutralized with 1M Tris pH 8.0. The OD at 280 nm of the dAbs wasmeasured and protein concentrations were determined using extinctioncoefficients calculated from the amino acid compositions of the dAbs.

The amino acid and nucleic acid sequences of the anti-human andanti-murine TGFRII dAb naive leads are given below.

Dom23h 802 amino acid sequence (SEQ ID NO: 1)EVQLLESGGGLVQPGGSLRLSCAASGFTFSEGTMWWVRQAPGKGLEWVSAILAAGSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKKRQERDGFDYWGQGTLVTVSSDom23h 802 nucleic acid sequence (SEQ ID NO: 39)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTAGTGAGGGGACGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGCTATTTTGGCTGCTGGTTCTAATACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAAAGAGGCAGGAGCGGGATGGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCDom23h 803 amino acid sequence (SEQ ID NO: 2)EVQLLESGGGLVQPGGSLRLSCAASGFTFSAGRMWWVRQAPGKGLEWVSAINRDGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHDDGHGNFDYWGQGTLVTVSSDom23h 803 nucleic acid sequence (SEQ ID NO: 40)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTAGTGCTGGGCGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGCGATTAATCGGGATGGTACTAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACATGATGATGGTCATGGTAAGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCDOM23h-813 amino acid sequence (SEQ ID NO: 3)EVQLLESGGGLVQPGGSLRLSCAASGSTFTDDRMWWVRQAPGKGLEWVSAIQPDGHTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEQDVKGSSSFDYWGQGTLVTVSSDOM23h-813 nucleic acid sequence (SEQ ID NO: 41)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATCCACCTTTACGGATGATAGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGCTATTCAGCCTGATGGTCATACGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGGAACAGGATGTTAAGGGGTCGTCTTCGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23h-815 amino acid sequence (SEQ ID NO: 4)EVQLLESGGGLVQPGGSLRLSCAASGFTFAEDRMWWVRQAPGKGLEWVSAIDPQGQHTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQSTGSATSDYWGQGTLVTVSSDOM23h-815 nucleic acid sequence (SEQ ID NO: 42)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGCGGAGGATCGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGCTATTGATCCTCAGGGTCAGCATACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACAGTCTACTGGGTCTGCTACGTCTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCDOM23h-828 amino acid sequence (SEQ ID NO: 5)EVQLLESGGGLVQPGGSLRLSCAASGFTFMSYRMWWVRQAPGKGLEWVSAISPSGSDTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQVVEYSRTHKGVFDYWGQGTLVTVSSDOM23h-828 nucleic acid sequence (SEQ ID NO: 43)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTATGAGTTATAGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGCTATTTCTCCGAGTGGTAGTGATACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACAGGTGGTGGAGTATTCGCGTACTCATAAGGGTGTGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23h-830 amino acid sequence (SEQ ID NO: 6)EVQLLESGGGLVQPGGFLRLSCAASGFTFEGYRMWWVRQAPGKGLEWVSAIDSLGDRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQGLTHQSPSTFDYWGQGTLVTVSSDOM23h-830 nucleic acid sequence (SEQ ID NO: 44)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTTCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGAGGGGTATAGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGCTATTGATTCTCTGGGTGATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACAGGGGCTTACGCATCAGTCTCCGAGTACGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23h-831 amino acid sequence (SEQ ID NO: 7)EVQLLESGGGLVQPGGSLRLSCAASGFTFEAYKMTWVRQAPGKGLEWVSYITPSGGQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYGSSFDYWGQGTLVTVSSDOM23h-831 nucleic acid sequence (SEQ ID NO: 45)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGAGGCGTATAAGATGACGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTGGAGTGGGTCTCATATATTACGCCGTCTGGTGGTCAGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAATATGGTTCGAGGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCDOM23h-840 amino acid sequence (SEQ ID NO: 8)EVQLLESGGGLVQPGGSLRLSCAASGFTFGDGRMWWVRQAPGKGLEWVSAIEGAGSDTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQASRNSPFDYWGQGTLVTVSSDOM23h-840 nucleic acid sequence (SEQ ID NO: 46)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGGGGATGGTCGTATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGCTATTGAGGGGGCGGGTTCGGATACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACAGGCGTCGCGGAATTCGCCGTTTGACTACTGGGGTCAGGGGACCCTGGTCACCGTCTCGAGCDOM23h-842 amino acid sequence (SEQ ID NO: 9)EVQLLESGGGLVQPGGSLRLSCAASGFTFDDSEMAWARQAPGKGLEWVSLIRRNGNATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVTKDRSVLFDYWGQGTLVTVSSDOM23h-842 nucleic acid sequence (SEQ ID NO: 47)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGATGATAGTGAGATGGCGTGGGCCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACTTATTCGGCGTAATGGTAATGCTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAGTTACGAAGGATCGTTCTGTGCTTTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23h-843 amino acid sequence (SEQ ID NO: 10)EVQLLESGGGLVQPGGSLRLSCAASGFTFDQDRMWWVRQAPGKGLEWVSAIESGGHRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQNESGRSGFDYWGQGTLVTVSSDOM23h-843 nucleic acid sequence (SEQ ID NO: 48)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGATCAGGATCGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGCTATTGAGAGTGGTGGTCATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACAGAATGAGTCGGGGCGTTCGGGGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCG AGCDOM23h-850 amino acid sequence (SEQ ID NO: 11)EVQLLESGGGLVQPGGSLRLSCAASGFTFDAARMWWARQAPGKGLEWVSAIADIGNTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQSGSEDHFDYWGQGTLVTVSSDOM23h-850 nucleic acid sequence (SEQ ID NO: 49)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGATGCGGCTAGGATGTGGTGGGCCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGCGATTGCGGATATTGGTAATACTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACAGTCTGGTTCGGAGGATCAGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCDOM23h-854 amino acid sequence (SEQ ID NO: 12)EVQLLESGGGLVQPGGSLRLSCAASGFTFAQDRMWWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQDLHGTSSLFDYWGQGTLVTVSSDOM23h-854 nucleic acid sequence (SEQ ID NO: 50)GAGGTGCAGCTGTTGGAGTCCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGCTCAGGATCGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACAGGATTTGCATGGTACTAGTTCTTTGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23h-855 amino acid sequence (SEQ ID NO: 13)EVQLLESGGGLVQPGGSLRLSCAASGFTFENTSMGWVRQAPGKGLEWVSRIDPKGSHTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQRELGKSHFDYWGQGTLVTVSSDOM23h-855 nucleic acid sequence (SEQ ID NO: 51)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGAGAATACGAGTATGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGTATTGATCCTAAGGGTAGTCATACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAATACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACAGCGTGAGTTGGGTAAGTCGCAGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCG AGCDOM23h-865 amino acid sequence (SEQ ID NO: 14)EVQLLESGGGLVQPGGSLRLSCAASGFTFRSYEMTWVRQAPGKGLEWVSKIDPSGRFTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGRTDLQLFDYWGQGTLVTVSSDOM23h-865 nucleic acid sequence (SEQ ID NO: 52)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTCGTAGTTATGAGATGACTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAAAGATTGATCCTTCGGGTCGIIIIACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAAGGTCGGACGGATCTTCAGCTTTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCDOM23h-866 amino acid sequence (SEQ ID NO: 15)EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYWMRWARQAPGKGLEWVSYITPKGDHTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAESLHNERVKHFDYWGQGTLVTVSSDOM23h-866 nucleic acid sequence (SEQ ID NO: 53)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCIIIICGAATTATTGGATGCGGTGGGCCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCATATATTACTCCTAAGGGTGATCATACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGGAATCGCTTCATAATGAGCGTGTTAAGCA GACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23h-874 amino acid sequence (SEQ ID NO: 16)EVQLLESGGGLVQPGGSLRLSCAASGFTFTSYRMWWVRQAPGKGLEWVSVIDSTGSATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQQAGSAMGEFDYWGQGTLVTVSSDOM23h-874 nucleic acid sequence (SEQ ID NO: 54)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTACTAGTTATCGTATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGTTATTGATTCTACTGGTTCGGCTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGCAGGCTGGGAGTGCGATGGGGGAGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23h-883 amino acid sequence (SEQ ID NO: 17)EVQLLESGGGLVQPGGSLRLSCAASGFTFVNYRMWWVRQAPGKGLEWVSAISGSGDKTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHGLSFDYWGQGTLVTVSSDOM23h-883 nucleic acid sequence (SEQ ID NO: 55)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGTTAATTATCGTATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGATAAGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACATGGGCTGTCGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCDOM23h-903 amino acid sequence (SEQ ID NO: 18)EVQLLESGGGLVQPGGSLRLSCAASGFTFNDMRMWWVRQAPGKGLEWVSVINADGNRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGLPFDYWGQGTLVTVSSDOM23h-903 nucleic acid sequence (SEQ ID NO: 56)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATGATATGAGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGTGATTAATGCTGATGGTAATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAAGATGGGCTGCCGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCDOM23m-4 amino acid sequence (SEQ ID NO: 19)EVQLLESGGGLVQPGGSLRLSCAASGFTFTTYGMGWVRQAPGKGLEWVSWIEKTGNKMADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAGRHIKVRSRDFDYWGQGTLVTVSSDOM23m-4 nucleic acid sequence (SEQ ID NO: 57)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTACGACTTATGGTATGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCATGGATTGAGAAGACGGGTAATAAGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAGCGGGGAGGCATATTAAGGTGCGTTCGAGGGAGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23m-29 amino acid sequence (SEQ ID NO: 20)EVQLLESGGGLVQPGGSLRLSCAASGFTFKRYSMGWVRQAPGKGLEWVSVINDLGSLTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGNISMVRPGSWFDYWGQGTLVTVSSDOM23m-29 nucleic acid sequence (SEQ ID NO: 58)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTAAGAGGTATTCTATGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGTTATTAATGATCTGGGTAGTTTGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAGGGAATATTAGTATGGTGAGGCCGGGGAGTTGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23m-32 amino acid sequence (SEQ ID NO: 21)EVQLLESGGGLVQPGGSLRLSCAASGFTFFEYPMGWVRQAPGKGLEWVSVISGDGQRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSHTGTVRHLETFDYWGQGTLVTVSSDOM23m-32 nucleic acid sequence (SEQ ID NO: 59)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTTTTGAGTATCCTATGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGTTATTAGTGGGGATGGTCAGCGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAAGTCATACGGGGACTGTGAGGCATCTGGAGACGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23m-62 amino acid sequence (SEQ ID NO: 22)EVQLLESGGGLVQPGGSLRLSCAASGFTFGQESMYWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGTRIKQGFDYWGQGTLVTVSSDOM23m-62 nucleic acid sequence (SEQ ID NO: 60)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGGTCAGGAGAGTATGTATTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAAGTGGTACGCGGATTAAGCAGGGGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCG AGCDOM23m-71 amino acid sequence (SEQ ID NO: 23)EVQLLESGGGLVQPGGSLRLSCAASGFTFMDYRMYWVRQAPGKGLEWVSGIDPTGLRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKIKWGEMGSYKTFDYWGQGTLVTVSSDOM23m-71 nucleic acid sequence (SEQ ID NO: 61)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTATGGATTATAGGATGTATTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGGGATTGATCCTACTGGTTTGCGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAATTAAGTGGGGGGAGATGGGGAGTTATAAGACGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23m-72 amino acid sequence (SEQ ID NO: 24)EVQLLESGGGLVQPGGSLRLSCAASGFTFMDYDMSWVRQAPGKGLEWVSMIREDGGKTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKARVPYRRGHRDNFDYWGQGTLVTVSSDOM23m-72 nucleic acid sequence (SEQ ID NO: 62)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTATGGATTATGATATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAATGATTCGTGAGGATGGTGGTAAGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAGCGAGGGTGCCTTATCGGCGTGGGCATAGGGATAAGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23m-81 amino acid sequence (SEQ ID NO: 25)EVQLLESGGGLVQPGGSLRLSCAASGFTFEPVIMGWVRQAPGKGLEWVSAIEARGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPGRHLSQDFDYWGQGTLVTVSSDOM23m-81 nucleic acid sequence (SEQ ID NO: 63)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCTTCCGGATTCACCTTTGAGCCGGTTATTATGGGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGCTATTGAGGCGCGGGGTGGGGGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACCTGGGCGGCATCTTAGTCAGGAGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCG AGCDOM23m-99 amino acid sequence (SEQ ID NO: 26)EVQLLESGGGLVQPGGSLRLSCAASGFTFDRYRMMWVRQAPGKGLEWVSTIDPAGMLTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRLASRSHFDYWGQGTLVTVSSDOM23m-99 nucleic acid sequence (SEQ ID NO: 64)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGATCGGTATCGTATGATGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAACGATTGATCCTGCTGGTATGCTTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAAGGCTGGCTTCGCGGAGTCAGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCDOM23m-101 amino acid sequence (SEQ ID NO: 27)EVQLLESGGGLVQPGGSLRLSCAASGFTFSEYDMAWVRQAPGKGLEWVSRIRSDGVRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRAKNGWFDYWGQGTLVTVSSDOM23m-101 nucleic acid sequence (SEQ ID NO: 65)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGCGCAGCCTCCGGATTCACCIIIICTGAGTATGATATGGCTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTTGAGTGGGTCTCACGGATTCGTTCTGATGGTGTTAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAGATCGTGCTAAGAATGGTTGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCDOM23h-352 amino acid sequence (SEQ ID NO: 28)EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYKMAWVRQAPGKGLEWVSLIFPNGVPTYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYSGQGRDFDYWGQGTLVTVSSDOM23h-352 nucleic acid sequence (SEQ ID NO: 66)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGATAAGTATAAGATGGCTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTGGAGTGGGTCTCACTTATTTTTCCGAATGGTGTTCCTACATACTACGCAAACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAATATAGTGGTCAGGGGCGGGAGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC

The CDRs as defined by Kabat of these anti-human and anti-murine TGFRIIdAb naive leads are shown in Tables 1 and 2, below, respectively.

TABLE 1  CDR Sequences of anti-human TGFβRII dAbs Clone CDR1 CDR2 CDR3DOM23h-802 SEGTMW AILAAGSNTYYADSVKG KRQERDGFDY (SEQ ID NO: 77)(SEQ ID NO: 113) (SEQ ID NO: 149) DOM23h-803 SAGRMW AINRDGTRTYYADSVKGHDDGHGNFDY (SEQ ID NO: 78) (SEQ ID NO: 114) (SEQ ID NO: 150) DOM23h-813TDDRMW AIQPDGHTTYYADSVKG EQDVKGSSSFDY (SEQ ID NO: 79) (SEQ ID NO: 115)(SEQ ID NO: 151) DOM23h-815 AEDRMW AIDPQGQHTYYADSVKG QSTGSATSDY(SEQ ID NO: 80) (SEQ ID NO: 116) (SEQ ID NO: 152) DOM23h-828 MSYRMWAISPSGSDTYYADSVKG QVVEYSRTHKGVFDY (SEQ ID NO: 81) (SEQ ID NO: 117)(SEQ ID NO: 153) DOM23h-830 EGYRMW AIDSLGDRTYYADSVKG QGLTHQSPSTFDY(SEQ ID NO: 82) (SEQ ID NO: 118) (SEQ ID NO: 154) DOM23h-831 EAYKMTYITPSGGQTYYADSVKG YGSSFDY (SEQ ID NO: 83) (SEQ ID NO: 119)(SEQ ID NO: 155) DOM23h-840 GDGRMW AIEGAGSDTYYADSVKG QASRNSPFDY(SEQ ID NO: 84) (SEQ ID NO: 120) (SEQ ID NO: 156) DOM23h-842 DDSEMALIRRNGNATYYADSVKG VTKDRSVLFDY (SEQ ID NO: 85) (SEQ ID NO: 121)(SEQ ID NO: 157) DOM23h-843 DQDRMW AIESGGHRTYYADSVKG QNESGRSGFDY(SEQ ID NO: 86) (SEQ ID NO: 122) (SEQ ID NO: 158) DOM23h-850 DAARMWAIADIGNTTYYADSVKG QSGSEDHFDY (SEQ ID NO: 87) (SEQ ID NO: 123)(SEQ ID NO: 159) DOM23h-854 AQDRMW AISGSGGSTYYADSVKG QDLHGTSSLFDY(SEQ ID NO: 88) (SEQ ID NO: 124) (SEQ ID NO: 160) DOM23h-855 ENTSMGRIDPKGSHTYYADSVKG QRELGKSHFDY (SEQ ID NO: 89) (SEQ ID NO: 125)(SEQ ID NO: 161) DOM23h-865 RSYEMT KIDPSGRFTYYADSVKG GRTDLQLFDY(SEQ ID NO: 90) (SEQ ID NO: 126) (SEQ ID NO: 162) DOM23h-866 SNYWMRYITPKGDHTYYADSVKG SLHNERVKHFDY (SEQ ID NO: 91) (SEQ ID NO: 127)(SEQ ID NO: 163) DOM23h-874 TSYRMW VIDSTGSATYYADSVKG QQAGSAMGEFDY(SEQ ID NO: 92) (SEQ ID NO: 128) (SEQ ID NO: 164) DOM23h-883 VNYRMWAISGSGDKTYYADSVKG HGLSFDY (SEQ ID NO: 93) (SEQ ID NO: 129)(SEQ ID NO: 165) DOM23h-903 NDMRMW VINADGNRTYYADSVKG DGLPFDY(SEQ ID NO: 94) (SEQ ID NO: 130) (SEQ ID NO: 166)

TABLE 2  CDR Sequences of anti-murine TGFβRII dAbs Clone CDR1 CDR2 CDR3DOM23m-4 TTYGMG WIEKTGNKTYYADSVKG AGRHIKVRSRDFDY (SEQ ID NO: 95)(SEQ ID NO: 131) (SEQ ID NO: 167) DOM23m-29 KRYSMG VINDLGSLTYYADSVKGGNISMVRPGSWFDY (SEQ ID NO: 96) (SEQ ID NO: 132) (SEQ ID NO: 168)DOM23m-32 FEYPMG VISGDGQRTYYADSVKG SHTGTVRHLETFDY (SEQ ID NO: 97)(SEQ ID NO: 133) (SEQ ID NO: 169) DOM23m-62 GQESMY AISGSGGSTYYADSVKGRSGTRIKQGFDY (SEQ ID NO: 98) (SEQ ID NO: 134) (SEQ ID NO: 170) DOM23m-71MDYRMY GIDPTGLRTYYADSVKG IKWGEMGSYKTFDY (SEQ ID NO: 99) (SEQ ID NO: 135)(SEQ ID NO: 171) DOM23m-72 MDYDMS MIREDGGKTYYADSVKGR ARVPYRRGHRDNFDY(SEQ ID NO: 100) (SEQ ID NO: 136) (SEQ ID NO: 172) DOM23m-81 EPVIMGAIEARGGGTYYADSVKG PGRHLSQDFDY (SEQ ID NO: 101) (SEQ ID NO: 137)(SEQ ID NO: 173) DOM23m-99 DRYRMM TIDPAGMLTYYADSVKG RLASRSHFDY(SEQ ID NO: 102) (SEQ ID NO: 138) (SEQ ID NO: 174) DOM23m-101 SEYDMARIRSDGVRTYYADSVKG DRAKNGWFDY (SEQ ID NO: 103) (SEQ ID NO: 139)(SEQ ID NO: 175) DOM23h-352 DKYKMA LIFPNGVPTYYANSVKG YSGQGRDFDY(SEQ ID NO: 104) (SEQ ID NO: 140) (SEQ ID NO: 176)

Example 2 DSC (Differential Scanning Calorimetry)—Naive Clones

dAbs thermal stability was determined using Differential Scanningcalorimetry (DSC). dAbs were dialysed overnight into PBS to a finalconcentration of 1 mg/ml. The dialysis buffer was used as a referencefor all samples. DSC measurements were performed using the GEHEALTHCARE™-MICROCAL™VP-DSC capillary cell microcalorimeter, at aheating rate of 180° C./hour. A typical scan range was from 20-90° C.for both the reference buffer and the protein sample. A rescan wasperformed each time in order to assess the extent of protein refoldingunder these experimental conditions. After each protein sample scan, thecapillary cell was cleaned with a solution of 5% DECON™(Fisher-Scientific) in water followed by a PBS scan. Resulting datatraces were analyzed using Origin 7.0 software. The DSC trace obtainedfrom the reference buffer scan was subtracted from that of the proteinsample scan. The precise molar concentration of the protein sample wasentered into the data analysis routine to yield values for meltingtemperature (Tm), enthalpy (ΔH) and Van't Hoff enthalpy (ΔHv) values.Data were fitted to a non-2-state model (N2M). The best fit was obtainedwith either 1 or 2 transition events. The Tm values obtained for thedAbs described in this patent range from 52.1° C. to 73.3° C. Tm valuesand percentage of refolding are shown in Table 3.

TABLE 3 DSC Apparent Tm ° C. 1-transition N2M 2-transition N2M dAb NameTm Tm1 Tm2 % refolding DOM23h-802 — 56.28 57.54 0 DOM23h-803 — 61.1964.59 23 DOM23h-813 52.11 — — 100 DOM23h-815 65.13 — — 93 DOM23h-828 —60.86 59.40 0 DOM23h-830 — 57.01 58.15 0 DOM23h-831 — 55.29 57.19 0DOM23h-840 63.70 — — 100 DOM23h-842 63.08 — — 27 DOM23h-843 60.15 — — 60DOM23h-850 58.27 — — 60 DOM23h-854 — 55.31 58.20 30 DOM23h-855 70.32 — —88 DOM23h-865 63.02 — — 0 DOM23h-866 — 52.88 55.77 18 DOM23h-874 — 58.8360.15 0 DOM23h-883 — 66.78 59.14 0 DOM23h-903 — 59.11 61.98 24 DOM23m-4— 57.1  61.3  0 DOM23m-29 68   — — 0 DOM23m-32 — 70.4  73.3  25DOM23m-62 — — — — DOM23m-71 63   — — 0 DOM23m-72 — — — — DOM23m-81 — — —— DOM23m-99 — 58.5  59   0 DOM23m-101 64   — — 30 DOM23m-352 66   — — 50

All molecules maintain tertiary structure up to at least 52° C. uponheating.

Example 3 SEC-MALS (Size Exclusion Chromatography withMulti-Angle-LASER-Light Scattering)—Naive Clones

To determine whether dAbs are monomeric or form higher order oligomersin solution, they were analyzed by SEC-MALLS (Size ExclusionChromatography with Multi-Angle-LASER-Light-Scattering). Agilent 1100series HPLC system with an autosampler and a UV detector (controlled byEmpower software) was connected to Wyatt Mini Dawn Treos (Laser LightScattering (LS) detector) and Wyatt Optilab rEX DRI (DifferentialRefractive Index (RI) detector). The detectors were connected in thefollowing order -UV-LS-RI. Both RI and LS instruments operate at awavelength of 658 nm; the UV signal was monitored at 280 nm and 220 nm.Domain antibodies (100 microliters injection at a concentration of 1mg/mL in PBS) were separated according to their hydrodynamic propertiesby size exclusion chromatography using a GE HEALTHCARE™ 10/300 Superdex75 column. The mobile phase was PBS plus 10% ethanol. The intensity ofthe scattered light while protein passed through the detector wasmeasured as a function of angle. This measurement taken together withthe protein concentration determined using the RI detector allowedcalculation of the molar mass using appropriate equations (integral partof the analysis software Astra v.5.3.4.14). All the dAbs describedherein have a monomeric content ranging from 65% to 98%. Data is shownin Table 4.

TABLE 4 dAb name Monomer by SEC-MALLS (%) DOM23h-802 92.5 DOM23h-80396.4 DOM23h-813 96.6 DOM23h-815 98 DOM23h-828 80 DOM23h-830 65DOM23h-831 72 DOM23h-840 91 DOM23h-842 91.6 DOM23h-843 90.2 DOM23h-85097.7 DOM23h-854 83.4 DOM23h-855 96.3 DOM23h-865 83 DOM23h-866 92.4DOM23h-874 92.6 DOM23h-883 93.5 DOM23h-903 96.5 DOM23m-4* 93 DOM23m-29*95 DOM23m-32* 92 DOM23m-62 Not determined DOM23m-71* 88 DOM23m-72 Notdetermined DOM23m-81 Not determined DOM23m-99 79 DOM23m-101 77.4DOM23m-352 93 *These dAbs were run using the same SEC-MALLS set up asdescribed above except that the HPLC used was a Shimadzu LC-20ADProminence system. These dAbs were also run on a Superdex75 column butthe mobile phase buffer was PBS.

The molecules listed in the tables 3 and 4 were chosen on the basis ofSolution State (propensity for monomer) content and Thermal stability.All molecules show a ≧65% propensity for monomerisation and maintaintertiary structure up to at least 52° C. upon heating.

Example 4 Assays for TGFbetaRII Inhibition (Naive Clones)

MC3T3-E1 Luciferase Assay—Method m1:

The MC3T3-E1 luciferase assay measures the ability of dAbs to inhibitTGFβ-induced expression of CAGA-luciferase in MC3T3-E1 cells. Threecopies of a TGFβ-responsive sequence motif, termed a CAGA box arepresent in the human PAI-1 promoter and specifically bind Smad3 and 4proteins. Cloning multiple copies of the CAGA box into a luciferasereporter construct confers TGFβ responsiveness to cells transfected withthe reporter system. This assay uses MC3T3-E1 cells (mouse osteoblasts)stably transfected with a [CAGA]₁₂-luciferase reporter construct(Dennler, et al. (1998) EMBO J. 17, 3091-3100).

Soluble dAbs were tested for their ability to block TGF-β1 signaling viathe Smad3/4 pathway.

The protocol used to generate the data which appears as method m1 intable 5, is as follows. Briefly, 2.5×10⁴ MC3T3-E1 cells per well inassay medium (RPMI medium (Gibco, Invitrogen Ltd, Paisley, UK), 10% heatinactivated foetal calf serum, and 1% penicillin/streptomycin) wereadded to a tissue culture 96 well plate (Nunc), followed by the dAb andTGF-β1 (final concentration 1 ng/ml) and incubated for six hours at 37°C., 5% CO₂. dAbs were dialysed into PBS prior to being tested in theassay. BRIGHTGLOW™ luciferase reagent (Promega, UK) was added to thewells and incubated at room temperature for two minutes to allow thecells to lyse, and the resulting luminescence measured on a luminometer.

The assay was performed multiple times to obtain an average and range ofmaximum % inhibitions values which are summarised in Table 5. Thismethod has been modified and is described Below.

Modified MC3T3-E1 Luciferase Assay—Method m2.

MC3T3-E1 cells were added to 96 well plates (Nunc 13610) at 1.25×10⁴ perwell in “plating medium” (MEM-Alpha+Ribonucleosides,+Deoxyribonucleosides (Invitrogen 22571), 5% Charcoal stripped FCS(Perbio Sciences UK Ltd; SH30068.03), 1/100 Sodium Pyruvate(Invitrogen11360), 250 μg/ml of Geneticin 50 mg/ml (Invitrogen,10131027), and incubated overnight at 37° C., 5% CO₂. The media from thecells was replaced with “assay media” (DMEM (Invitrogen 31966021,) 25 mMHepes (Invitrogen)), and purified dAbs in PBS at 4× final assayconcentration were titrated in “assay media” and added to the cellplates, followed by TGF-β1 (R&D, 240B) at 4× the EC80. The plates wereincubated for six hours at 37° C., 5% CO₂. STEADYLITE™ luciferasereagent (PerkinElmer 6016987) was added to the wells and incubated atroom temperature for 30 minutes, and the resulting luminescence measuredon a the ENVISION™ plate reader.

Each dAb was titrated in duplicate in an assay and a maximum %inhibition determined (n=2). The assay was performed multiple times toobtain an average and range of maximum % inhibitions values which aresummarised in Table 5. The assay QC parameters were met; in-house smallmolecule showing an IC50 range from 100 to 900 nM for the mouse assays.Also, the robust Z factors were greater than 0.4 and the TGF-β EC80 waswithin 6 fold of the concentration added to the assay.

A549 IL-11 Release Assay—h1

The A549 Interleukin-11 (IL-11) release assay measures the ability ofdAbs to inhibit human TGF-β1 stimulated IL-11 release from A549 cells.TGF-β1 binds directly to TGF-βRII and induces the assembly of theTGF-βRI/II complex. TGF-βRI is phosphorylated and is able to signalthrough several pathways including the Smad4 pathway. Activation of theSmad4 pathway results in the release of IL-11. The IL-11 is secretedinto the cell supernatant and is then measured by colourmetric ELISA.

Soluble dAbs were tested for their ability to block TGF-β1 signallingvia the Smad4 pathway. Briefly, 1×10⁵ A549 cells per well in “assaymedium” (DMEM high glucose medium (Gibco™, Invitrogen Ltd, Paisley, UK),10% heat inactivated foetal calf serum (PAA, Austria), 10 mM HEPES(Sigma, UK) and 1% penicillin/streptomycin (PAA, Austria)) were added toa tissue culture 96 well plate (Nunc), followed by the dAb and TGF-β1(final concentration 3 ng/ml) (R&D Systems, Abingdon, UK) and incubatedovernight at 37° C., 5% CO₂. dAbs were dialysed into PBS prior to beingassayed. The concentration of IL-11 released into the supernatant wasmeasured using a Human IL-11 DUOSET™ (R&D systems, Abingdon, UK), inaccordance with the manufacturer's instructions.

The A549 IL-11 release assay is referred to in tables 5 and 6 as assaymethod h1. The assay was performed multiple times to obtain an averageand range of maximum % inhibitions values which are summarised in Table5. The assay QC parameters were met; in-house small molecule showing anIC50 range from 50 to 500 nM for the human assays.

SBE-bla HEK 293T Cell Sensor Assay—h2:

Members of the Smad family of signal transduction molecules arecomponents of an intracellular pathway that transmits TGF-β signals fromthe cell surface to nucleus. TGF-β1 binds directly to TGF-βRII andinduces the assembly of the TGF-βRI/II complex. Smad2 and Smad3 are thenphosphorylated by TGF-βRI, and subsequently form a heteromeric complexwith the co-smad family member Smad4. These complexes are translocatedto the nucleus where they bind DNA and regulate gene transcription.

Cell Sensor SBE-bla HEK 293T cells contain a beta-lactamase reportergene under control of the Smad binding element (SBE) which was stablyintegrated into HEK 293T cells (Invitrogen, UK). The cells areresponsive to TGF-βI and can be used to detect agonists/antagonists ofthe Smad2/3 signaling pathway.

Soluble dAbs were tested for their ability to block TGF-β1 signaling viathis pathway following the method below, which was based on an optimisedmethod from Invitrogen, UK, (cell line K1108).

The assay was performed direct from frozen cells which had been grownfor at least 4 passages in growth media (DMEM high glucose, Invitrogen21068028, 10% Dialysed U.S. FBS. Invitrogen 26400-044, 0.1 mM (1/100)Non essential amino acids. Invitrogen 11140-050, 25 mM (1/40) HEPESbuffer. Sigma H0887, 1 mM (1/100) Sodium pyruvate. Invitrogen 11360-070,1% GLUTAMAX™. (200 mM Invitrogen 35050038), 5 μg/ml of Blasticidin.Invitrogen R21001) and frozen in house (at 4×10⁷/ml). The cells wereplated at 20,000 cells per well in cell culture plates (Costar 3712) inplating media (as above with 1% FCS and no blasticidin). Afterincubating the cells overnight, the purified dAbs were diluted in “assaymedia” (DMEM (Invitrogen 31966021,) 25 mM Hepes (Invitrogen) and addedto the cells at 4× final assay concentration. After a 1 hour incubationat 37° C., TGF-β (R&D Systems; 240B) was added at 4×EC80 and incubatedfor a further 5 hours. The LIVEBLAZER™ substrate (Invitrogen K1030), wasmade up according to the manufacturer's instructions and added at 8× thevolume. The plates were incubated in the dark at room temperature for 16hours and read on the ENVISION™ plate reader according to the Invitrogenprotocol.

The SBE-bla HEK CELLSENSOR™ assay is referred to in tables 5 and 6 asmethod h2. Each dAb was titrated in duplicate in an assay and an IC50determined and maximum % inhibition determined (n=2). Due to thedifficulty of obtaining full curves in the mouse assay, only %inhibitions are quoted in table 5. The assay was performed multipletimes to obtain an average and a range of values which are summarised inTables 5 and 6. The arithmetric mean IC50 was calculated using pIC50's(−log of IC50), and the range calculated adding and subtracting the logstandard deviation from mean pIC50, and then transforming back to IC50.The assay QC parameters were met; in-house small molecule showing anIC50 range from 50 to 500 nM for the human assays. Also, the robust Zfactors were greater than 0.4 and the TGF-β EC80 was within 6 fold ofthe concentration added to the assay

The results are shown in Tables 5 and 6.

TABLE 5 Cell Functional assay data for mouse specific clones plus VHDummy dAb. Human IL-11 release (h1) or SBE-bla HEK Mouse 3T3 cell assayCELLSENSOR ™ assay (h2) Assay max % inhibition Assay max % inhibitionMethod Average SD range n Method Average SD range n DOM23m-04 m1 73.38.4 68.8-83   3 h1 70.7 6.4 67-78 3 DOM23m-04 h2 69.0 1 DOM23m-29 m154.2 5.2 50.5-57.9 2 DOM23m-32 m1 39.7 4.0 36.9-42.5 2 DOM23m-62 m1 78.61 h1 79.0 1 DOM23m-71 m1 44.9 9.3 38.3-51.4 2 h1 −2.0 1 DOM23m-72 m117.5 24.7   0-34.9 2 h1 1.7 1 DOM23m-81 m2 30.3 11.5 21-47 4 DOM23m-99m2 46.5 28.0 26.7-93.5 6 DOM23m-101 m2 48.0 19 22.0-74.1 12 h2 59.8 27.017-81 5 DOM23h-352 m2 48.0 23.9 16.8-78.9 16 h2 46.7 36.5 5.7-86  5VHDUM-2 m2 21.4 13.2   21-33.9 15 h2 46.0 29.6 17-84 6 VHDUM-2 m1 22.5 022.5 2

TABLE 6 Cell Functional data for human specific clones plus VH DummydAb. IC50 nM Assay IC50 range method dAb Mean (+/− log SD) n h2DOM23h-802 >11062 6592-18562 6 h2 DOM23h-803 >11619 5890-22922 6 h2DOM23h-813 >9328 4301-20230 6 h2 DOM23h-815 7122 3026-16764 4 h2DOM23h-828 9899.07 4441-22065 4 h2 DOM23h 830 6299 5442-7291  4 h2DOM23h-831 >3126  534-18291 8 h2 DOM23h 840 2915  650-13081 7 h2 DOM23h842 2042 2223-18704 4 h2 DOM23h-843 >9007 3396-23894 8 h2 DOM23h-8505350 2358-12137 6 h2 DOM23h-854 >9551 3085-29569 8 h2 DOM23h-855 >44671088-18339 8 h2 DOM23h 865 5559 1070-28893 4 h2 DOM23h 866 >1762 195-15900 6 h2 DOM23h 874 >925  89-9591 6 h2 DOM23h 883 10123  60-173446 h2 DOM23h 903 1048 492-223  5 h2 VHDummy-2 >25119 25000-50000  12

The mouse clones were selected on the basis that they showed greaterthan 40% neutralisation of TGF-β in several assays. The only exceptionto this was DOM23m-72. The clones also showed good neutralisation curves(data not shown). The human clones were selected on the basis that theaverage IC50's were less than 15 μM.

Example 5 Error Prone Affinity Maturation of Naive Clones (from Example1)

Error-prone mutagenesis was performed to improve the affinity of thedAbs identified as active with suitable biophysical characteristics(described above).

Phage Library Construction: Error prone libraries of DOM23h-843,DOM23h-850, DOM23h-854, DOM23h-855, DOM23h-865, DOM23h-866, DOM23h-874,DOM23h-883, DOM23h-439 and DOM23h-903, were made using GENEMORPH™ IIRandom Mutagenesis kit (Stratagene, Cat No 200550). The target dAb geneswere amplified by PCR using Taq DNA polymerase and oligonucleotidesDOM008 (5′-AGCGGATAACAATTTCACACAGGA-3′ (SEQ ID NO:185)) and DOM009(5′-CGCCAGGGTTTTCCCAGTCACGAC-3′ (SEQ ID NO:186)), followed byre-amplification of the diluted PCR product with oligonucleotides DOM172(5′ TTGCAGGCGTGGCAACAGCG-3′ (SEQ ID NO:187)) and DOM173(5f-CACGACGTTGTAAAACGACGGCC-3′ (SEQ ID NO:188)), and MUTAZYME™ II DNApolymerase, according to manufacturer's instructions. This PCR productwas further amplified using Taq DNA polymerase and oligonucleotidesDOM172 and DOM173, to increase the DNA product yield. The PCR productwas digested with Sal I and Not I restriction endonucleases. Undigestedproduct and digested ends were removed from the digested product usingstreptavidin beads (Dynal Biotech, UK). For the anti-human error proneselections digested product was ligated into pDOM4 phage vector digestedwith Sal I and Not I restriction endonucleases and used to transform E.coli TB1 cells. The transformed cells were plated on 2×TY agarsupplemented with 15 μg/ml tetracycline, yielding library sizes of>1×10⁷ transformants.

Human TGFbetaRII specific dAb Error-prone selections: Three rounds ofselection were performed with the DOM23h-843, DOM23h-850, DOM23h-854,DOM23h-855, DOM23h-865, DOM23h-866, DOM23h-874, DOM23h-883, DOM23h-903,and DOM23h-439 libraries. Round one was performed using 1 nMbiotinylated human TGFbetaRII/Fc (N13241-57). Two different methods werefollowed for rounds two and three, method 1 using the dimericTGFbetaRII/Fc form of the antigen and method two using the soluble,monomeric form of TGFbetaRII. Method 1: Round two was performed with 1nM biotinylated human TGFbetaRII/Fc with 1 uM non-biotinylated humanTGFbetaRII/Fc competitor. Round three was performed with 100 pMbiotinylated human TGFbetaRII/Fc with 1 uM non-biotinylated humanTGFbetaRII/Fc (N12717-4). Method 2: Round two was performed with 1 nMbiotinylated human TGFbetaRII with 1 uM non-biotinylated humanTGFbetaRII competitor. Round three was performed with 100 pMbiotinylated human TGFbetaRII with 1 uM non-biotinylated humanTGFbetaRII competitor.

Second and third round selection outputs were subcloned into the pDOM13vector, as described above. Individual clones were picked and expressedin 96 well plates at 850 rpm, 37° C. for 24 hours, 90% humidity in 0.5ml/well overnight express auto-induction medium supplemented with 100μg/ml carbenicillin. Plates were then centrifuged at 1800 g for 10minutes. Supernatants were diluted either 1/5 or 1/2 in HBS-EP bufferand screened on BIACORE™ for binding to biotinylated human TGF-β RII/Fc(SA chip coated with 1000 Ru biotinylated hRII-Fc in accordance with themanufacturer's recommendations) (BIACORE™, GE HEALTHCARE™). Samples wererun on BIACORE™ at a flow rate of 50 μl/min. Clones that bound with ahigh number of resonance units (RUs) or with an improved off-ratecompared to the parent clone were expressed in 50 ml overnight expressautoinduction medium at 30° C. for 48 to 72 hours and centrifuged at4,600 rpm for 30 minutes. The supernatants were incubated overnight at4° C. with Streamline-protein A beads. The beads were then packed intodrip columns, washed with 5 column volumes of 2×PBS, followed by one bedvolume of 10 mM Tris-HCl pH 7.4 and bound dAbs were eluted in 0.1 Mglycine-HCl, pH 2.0 and neutralised with 1 M Tris-HCl, pH 8.0. The OD at280 nm of the dAbs was measured and protein concentrations weredetermined using extinction coefficients calculated from the amino acidcompositions of the dAbs.

In vitro analysis of off rate improved error prone selections: PurifieddAbs were subjected to the same tests as those from the naiveselections, namely, Biacore, SBE-bla HEK 293T Cell Sensor assay (h2),DSC, and SEC-MALS. Examples of clones improved over parent are shown intable 6A. IC50 values are a mean of ‘n’ number of experiments.

TABLE 6A On-rate Off-rate Affinity ka Fold kd Fold KD Fold Mean IC50DOM23h ka1 (1/Ms) kd1 (1/s) KD improvement improvement improvement (nM)*439 2.08E+06 5.02E−02 2.42E−08    3570 (3) 439-20 4.43E+06 3.54E−037.99E−10 2.1 14.2 30.3     48 (10) 843 9.05E+05 3.43E−01 3.78E−07   1947 (3) 843-13 5.11E+06 2.11E−02 4.13E−09 5.6 16.2 91.7    540 (4)855 3.35E+05 3.15E−01 9.41E−07 >25000 (3) 855-21 1.86E+06 3.36E−021.80E−08 5.6  9.4 52.3   18580 (6) *number of experiments forcalculation of mean IC50s provided in parenthesisAffinity Matured Sequences

DOM23h-855-21 nucleic acid sequence (SEQ ID NO: 203)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGAGAATACGAGTATGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGTATTGATCCTAAGGGTAGTCATACATACTACACAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAATACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACAGCGTGAGTTGGGTAAGTCGTAGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCDOM23h-855-21 amino acid sequence (SEQ ID NO: 204)EVQLLESGGGLVQPGGSLRLSCAASGFTFENTSMGWVRQAPGKGLEWVSRIDPKGSHTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQRELGKSYFDYWGQGTLVTVSSDOM23h-843-13 nucleic acid sequence (SEQ ID NO: 205)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCCTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGATCAGGATCGGATGTGGTGGGTCCGCCAGGCCCCAGGGAAGGGTCTAGAGTGGGTCTCAGCTATTGAGAGTGGTGGTCATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAATCAGAATAAGTCGGGGCGTTCGGGGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCDOM23h-843-13 amino acid sequence (SEQ ID NO: 206)EVQLLESGGGLVQPGGSLRLSCAASGFTFDQDRMWWVRQAPGKGLEWVSAIESGGHRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANQNKSGRSGFDYWGQGTLVTVSSDOM23h-439-20 nucleic acid sequence (SEQ ID NO: 207)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTGTCTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACGGCATGCGGCTGGGGTTTCGGGTACTTATTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23h-439-20 amino acid sequence (SEQ ID NO: 208)EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFVSRIDSPGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRHAAGVSGTYFDYWGQGTLVTVSS

Example 6 Affinity Maturation of DOM23h-271-7 Lineage

DOM23h-271 amino acid sequence (SEQ ID NO: 199)EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQIPGRKWTANSRFDYWGQGTLVTVSSDOM23h-271 nucleic acid sequence (SEQ ID NO: 200)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTAATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGATTCCGGGGCGTAAGTGGACTGCTAATTCGCGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23h-271-7 amino acid sequence (SEQ ID NO: 201)EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQIPGRKWTANSRFDYWGQGTLVTVSSDOM23h-271-7 nucleic acid sequence  (SEQ ID NO: 202)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTAATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGATTCCGGGGCGTAAGTGGACTGCTAATTCGCGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC

Domain antibody DOM23h-271(SEQ ID No:199) had been isolated from thephage libraries in a previous selection campaign and variantDOM23h-271-7 (SEQ ID NO:201) was isolated following error prone affinitymaturation, both as described in WO 2011/012609DOM23h-271-7 (SEQ IDNO:201) was selected for further affinity maturation based on itsbinding kinetics, sequence and biophysical behaviour. Affinitymaturation was performed using degenerative mutagenesis to re-diversifythe CDRs, and improved leads were identified using DNA display or phagedisplay. Two types of libraries were constructed to re-diversify theCDRs, and these are referred to as triplet and doped libraries. To makethe triplet libraries oligonucleotide primers were designed to covereach CDR, and within each primer the codons for three amino acids werereplaced with NNS codons, so that three positions were diversified.Multiple oligonucleotides were used to cover all targeted amino acidswithin each CDR: 2 for CDR1, 3 for CDR2 and 6 for CDR3. Complementaryoligonucleotide primers were used to amplify a sequence fragmentcontaining each mutated CDR, and also an overlapping sequence fragmentcovering the rest of the dAb coding sequence. These fragments were mixedand assembled by splice extension overlap PCR to produce the full lengthdAb coding sequence. This product was PCR amplified using primers DOM172(SEQ ID NO:187) and DOM173 (SEQ ID NO:188), digested with SalI and NotI,and ligated into similarly cut pDOM4 (described above) for phageselections, or pIE2A2 (described in WO2006018650) for DNA Displayselections. The doped libraries were constructed using a similar method,essentially as described in WO2006018650. A single degenerateoligonucleotide primer was used to cover all mutations within each CDR.Within each primer the amino acids to be diversified were specifiedusing degenerate codons to specify multiple amino acids. Five aminoacids were diversified in CDR1, 7 in CDR2, and 13 in CDR3. In theprimers the following degenerate coding is used: ‘a’=91% A+3% T+3% G+3%C; ‘g’=91% G+3% T+3% C+3% A; ‘c’=91% C+3% T+3% G+3% A; ‘t’=91% T+3% A+3%G+3% C; ‘S’=50% G and 50% C. Capital letters indicate 100% of thespecified nucleotide. The primers used were:

271-7R1deg CDR1 (SEQ ID NO: 189)(GCAGCCTCCGGATTCACCTTTacSgaStatagSATGtgSTGGGTCCGCCAGGCTCCGGGG);271-7R2deg CDR2 (SEQ ID NO: 190)(GGGTCTCGAGTGGGTCTCAgcSATTgaSccSatSggSaaScgSACATACTACGCAGACTCCGTG);271-7R3deg CDR3: (SEQ ID NO: 191)(GCGGTATATTACTGTGCGAAAcaSatSccSggScgSaaStgSacSgcSaaStcScgSttSGACTACTGGGGTCAGGG).

The degenerate library primers were used in the same way as the tripletprimers. Each diversified CDR was amplified separately and then combinedwith a parental sequence fragment using splice extension overlap. Thefragments were subcloned to pDOM4 and pIE2A2 using SalI and NotI.

DNA Display

Selections were performed using in vitro compartmentalisation inemulsions and DNA display using the scArc DNA binding proteinessentially as described in WO2006018650. Briefly, TGFbRII-FC antigenwas biotinylated using a 5:1 molar ratio of Biotin and the EZ-LINK™Sulpho-NHS-LC-Biotin kit (Thermo #21327). A DNA fragment containing theArc operator sequences and expression cassette containing thediversified dAb library was PCR amplified from the pIE2A2 vector usingflanking primers. The product was purified from an eGel (Invitrogen) anddiluted to 1.7 or 0.85 nM in 1 mg/ml BSA. For selection of improvedbinders the doped and Triplet libraries were processed separately underslightly different conditions. The Triplet CDR libraries were combinedto give pooled CDR 1, 2 or 3 libraries. Ten rounds of selection wereused for each type of library. For both methods, after 2 selectioncycles, the diversified CDR's were amplified and recombined by spliceoverlap extension PCR to produce a 4th library with mutations in all 3CDRs.

For the doped libraries 5×10⁸ copies of DNA were mixed with 50 ul ofEXPRESSWAY™ In vitro translation mix (Invitrogen). Each reactioncontained 10.0 μl SLYD™ extract; 10.0 μl 2.5× reaction buffer; 12.5 μl2× feed buffer; 1.0 μl Methionine (75 mM); 1.26 μl Amino Acid mix (50mM); 15 μl H₂O; 0.5 μl T7 Polymerase; 0.25 μl anti-HA mAb 3F10 (Roche,cat. 1 867 423); and 1.5 μl Glutathione (100 mM) (Sigma). This was addedto 800 μl of hydrophobic phase (4.5% SPAN™-80, (Fluka)+0.5% Triton X-100(Sigma) in Light white mineral oil (Sigma)) in a 4 ml glass vial(CHROMACOL™ 4SV P837) and stirred at 2000 rpm for 4-5 minutes. The tubeswere sealed and incubated for 3 hours at 30° C. All subsequent stepswere done at room temperature. To extract the DNA-protein complexes 200μl of C+ buffer (10 mM Tris, 0.1 M KCl, 0.05% TWEEN™-20, 5 mM MgCl₂, 1%BSA, pH 7.4) and 500 μl of Hexane was added to the vial, mixed andtransferred to a microtube and centrifuged at 13000 g for 1 minute. Theorganic phase was removed and the aqueous phase re-extracted with 800 μlof Hexane 3-5 more times until the interface was almost clear. For thefirst 5 rounds of selections the biotinylated TGFbRII-FC was pre-boundto Streptavidin DYNAbeads™ (Invitrogen) and added to the extractedcomplexes to give an antigen concentration equivalent to 40, 40, 10, 5and 5 nM antigen (rounds 1, 2, 3, 4 and 5 respectively). T1 beads wereused for selections 1-3, and C1 for selections 4 and 5. After 30 minutesincubation the beads were washed 3-5 times with C+ buffer. The DNAcomplexes remaining bound to the beads were then recovered by PCR withflanking primers. Selection rounds 6-10 were referred to as ‘soluble’selections, where the Biotinylated TGFbRII-FC was added directly to thecomplexes after extraction to give a concentration of 5, 5, 4, 5 and 5nM (rounds 6, 7, 8, 9 and 10 respectively), and incubated for 30 minutesto allow binding to be established. Non-biotinylated TGFbRII-FC was thenadded as a competitor to 74, 750, 400, 250 and 250 nM (rounds 6, 7, 8, 9and 10 respectively), and incubated for 15, 15, 30, 30 and 30 minutes(rounds 6, 7, 8, 9 and 10 respectively). In rounds 9 and 10 a doublestranded oligonucleotide containing the ARC operator sequence wasincluded at 50 nM in the C+ buffer used in the hexane extractions toreduce cross-reactions between any non-complexed DNA and excess proteinreleased from the emulsions. After the competition period 10 μl of C1Streptavidin DYNAbeads™ were added. After 10 minutes the beads werewashed 5 times with Buffer C+ and the bound complexes recovered by PCRwith flanking primers as previously described. The PCR product waspurified on an eGel and used for the next selection cycle. Following the10th selection the recovered product was cut with SalI and NotI enzymes,and cloned into similarly cut pDOM13 for expression.

The triplet libraries were selected using a similar method, except that1×10⁹ DNA copies were used in the first round selection, and 5×10⁸thereafter. Also, the incubation time for protein expression in theemulsion was reduced to 2 hours. Soluble Biotinylated TGFbRII FC wasused in all ten selection rounds at 25, 10, 5, 5, 5, 5, 2.5, 2.5, 2.5,2.5, 2.5, 2.5 nM respectively. The Biotinylated target was incubatedwith the extracted complexes for 30 minutes. In selection rounds 5-10the non-biotinylated TGFbRII-FC competitor was added to a finalconcentration of 250 nM for 15, 30, 60, 60, 75, and 90 minutesrespectively before addition of C1 streptavidin DYNAbeads™. In round 5competition was at room temperature, but from round 6 the competitiontemperature was increased to 30° C. The Arc Operator decoy oligo wasincluded in selection rounds 1-4 to reduce cross complexing of defectiveDNA.

Following selections the dAb encoding inserts were excised from the DNAdisplay expression cassettes using SalI and NotI, and cloned into thepDOM13 bacterial expression vector. The dAbs were sequenced andexpressed in TB ONEX™ medium and supernatants were screened by BIACORE™to identify clones with improved off-rates when compared to parent.Clones with improved off-rates were expressed and purified, and wereassessed for affinity by BIACORE™ and potency in the cell sensor assay.Clones giving poor kinetic profiles, containing unfavourable sequencemotifs, or giving very low yields were not pursued. Three were selectedto be of further interest. Clones DOM23h-271-21 (SEQ ID NO:29) andDOM23h-271-22 (SEQ ID NO:30) were isolated from doped libraryselections. Clone DOM23h-271-27 (SEQ ID NO:31) was isolated from atriplet library selection. The affinity of the selected clones for humanTGFbRII-FC is shown in table 7.

TABLE 7 Ka(M⁻¹ · s⁻¹) Kd (s⁻¹) KD (nM) DOM23h-271-7* 5.37E+6 5.10E−29.49 DOM23h-271-21 3.21E+6 4.72E−4 0.147 DOM23h-271-22 3.22E+6 9.19E−40.286 DOM23h-271-27 2.17E+6 1.26E−3 0.578 *N.B. values in the abovetable are for ranking purposes only since the fitting for DOM23h-271-7to the 1:1 model was poor, although the affinity matured samples fittedwell to this model.Phage Display:

Triplet or doped libraries in separate CDR1, CDR2 and CDR3 pools weresubjected to rounds of phage selection as described above against eitherbiotinylated human TGF-β RII/Fc antigen over 4 rounds in concentrationsof 10 nM, 1 nM, 100 pM and 20 pM respectively, or two rounds ofselection using 20 pM followed by 2 pM antigen. Inserts from phageselections were cloned into the pDOM10 expression vector andsupernatants with off rates improved over parent were selected forfurther study. Domain antibodies were expressed and purified theiraffinity and bioactivity against human TGF-β RII/Fc antigen tested onthe BIACORE™ T100 and in the Cell sensor assay described above (data notshown). The affinity of the selected clones for human TGFbRII-FC isshown in table 8.

TABLE 8 Sample Ka (M−1 · s−1) Kd (s−1) KD (M) 271-101 3.73E+06 0.020145.40E−09 271-102 9.35E+06 0.01531 1.64E−09 271-105a* 3.29E+06 0.007472.27E−09 271-105b* 3.07E+06 0.007977 2.60E−09 271-106a* 7.39E+06 0.023333.16E−09 271-106b* 6.77E+06 0.01947 2.88E−09 271-114 1.04E+07 0.070846.80E−09 271-7a* 3.04E+06 0.04193 1.38E−08 271-7b* 2.42E+06 0.044481.84E−08 *The designation “a” and “b” refer to separate supernatantsresulting from different colonies of the numbered clones.

The sequence of the selected clones with improved activity wasdetermined and the full sequences and CDR sequences are shown below.

DOM23h-271-21 amino acid sequence  (SEQ ID NO: 29)EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQMPGRKWTAKFRWDYWGQGTLVIVSSDOM23h-271-21 nucleic acid sequence  (SEQ ID NO: 67)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTACCGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTAATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGATGCCGGGCCGGAAGTGGACGGCCAAGTTCCGCTGGGACTACTGGGGTCAGGGAACCCTGGTCATCGTCTCGAGC DOM23h-271-22 amino acid sequence  (SEQ ID NO: 30)EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQMPGQKWMAKSRFDYWGQGTLVTVSSDOM23h-271-22 nucleic acid sequence  (SEQ ID NO: 68)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTAATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGATGCCCGGCCAGAAGTGGATGGCCAAGTCCCGCTTCGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23h-271-27 amino acid sequence  (SEQ ID NO: 31)EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGQKMADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQIPGRKWTANSRFDYWGQGTLVIVSSDOM23h-271-27 nucleic acid sequence  (SEQ ID NO: 69)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTCAGAAGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGATTCCGGGGCGTAAGTGGACTGCTAATTCGCGGTTTGACTACTGGGGTCAGGGAACCCTGGTCATCGTCTCGAGC DOM23h-271-101 amino acid sequence  (SEQ ID NO: 32)EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQIPGRKWTANGRKDYWGQGTLVTVSSDOM23h-271-101 nucleic acid sequence  (SEQ ID NO: 70)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTAATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGATTCCGGGGCGTAAGTGGACTGCTAATGGTCGTAAGGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23h-271-102 amino acid sequence  (SEQ ID NO: 33)EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQIPGRKWTANSRFDYWGQGTLVTVSSDOM23h-271-102 nucleic acid sequence  (SEQ ID NO: 71)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGATTCCGGGGCGTAAGTGGACTGCTAATTCGCGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23h-271-105 amino acid sequence  (SEQ ID NO: 34)EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQIPGQRWTGNSRFDYWGQGTL VTVSSDOM23h-271-105 nucleic acid sequence  (SEQ ID NO: 72)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTAATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGATTCCGGGGCAGCGGTGGACTGGTAATTCGCGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23h-271-106 amino acid sequence  (SEQ ID NO: 35)EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQFPGRKWTANSRSDYWGQGTLVTVSSDOM23h-271-106 nucleic acid sequence  (SEQ ID NO: 73)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTAATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGTTTCCGGGGCGTAAGTGGACTGCTAATTCGCGGTCTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23h-271-114 amino acid sequence  (SEQ ID NO: 36)EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQIPGRKGTANSRFDYWGQGTLVTVSSDOM23h-271-114 nucleic acid sequence  (SEQ ID NO: 74)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTAATCGTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGATTCCGGGGCGTAAGGGAACTGCTAATTCGCGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC

TABLE 9CDR sequences of 271 affinity matured clones with improved activityCDR1 (Kabat 26-35) CDR2 (Kabat 50-65) CDR3 (Kabat 95-102) DOM23h-GFTFTEYRMW AIEPIGNRTYYADSVKG QMPGRKWTAKFRWDY 271-21 (SEQ ID NO: 105)(SEQ ID NO: 141) (SEQ ID NO: 177) DOM23h- GFTFTEYRMW AIEPIGNRTYYADSVKGQMPGQKWMAKSRFDY 271-22 (SEQ ID NO: 106) (SEQ ID NO: 142)(SEQ ID NO: 178) DOM23h- GFTFTEYRMW AIEPIGQKTYYADSVKG QIPGRKWTANSRFDY271-27 (SEQ ID NO: 107) (SEQ ID NO: 143) (SEQ ID NO: 179) DOM23h-GFTFTEYRMW AIEPIGNRTYYADSVKG QIPGRKWTANGRKDY 271-101 (SEQ ID NO: 108)(SEQ ID NO: 144) (SEQ ID NO: 180) DOM23h- GSTFTEYRMW AIEPIGHRTYYADSVKGQIPGRKWTANSRFDY 271-102 (SEQ ID NO: 109) (SEQ ID NO: 145)(SEQ ID NO: 181) DOM23h- GFTFTEYRMW AIEPIGNRTYYADSVKG QIPGQRWTGNSRFDY271-105 (SEQ ID NO: 110) (SEQ ID NO: 146) (SEQ ID NO: 182) DOM23h-GFTFTEYRMW AIEPIGNRTYYADSVKG QFPGRKWTANSRSDY 271-106 (SEQ ID NO: 111)(SEQ ID NO: 147) (SEQ ID NO: 183) DOM23h- GFTFTEYRMW AIEPIGNRTYYADSVKGQIPGRKGTANSRFDY 271-114 (SEQ ID NO: 112) (SEQ ID NO: 148)(SEQ ID NO: 184) N.B CDR2 and CDR3 are as defined by Kabat. CDR1 isdefined by a combination of the Kabat and Chothia methods.Generic Method for Binding Kinetics—T100

BIACORE™ analysis was carried out using a capture surface on a CM4 chip.Anti-human IgG was used as the capturing agent and coupled to a CM4biosensor chip by primary amine coupling. The Antigen molecule fused tothe human Fc was captured on this immobilised surface to a level from250 to 300 resonance units and defined concentrations of Domainantibodies diluted in running buffer were passed over this capturedsurface. An injection of buffer over the captured antigen surface wasused for double referencing. The captured surface was regenerated, aftereach domain antibody injection using 3M magnesium chloride solution; theregeneration removed the captured antigen but did not significantlyaffect the ability of the surface to capture antigen in a subsequentcycle. All runs were carried out at 25° C. using HBS-EP buffer asrunning buffer. Data were generated using the BIACORE™ T100 and fittedto the 1:1 binding model inherent to the software. When non-specificbinding was seen at the top concentration, the binding curve at thisconcentration was removed from the analysis set.

Further Diversification of CDR3

The DOM23h-271-7 derivatives with the highest affinity containedmethionines in position 96 and 100B. These positions, along withpositions 99, 100D, 100E and 100G were diversified using NNK mutagenesisto determine whether substitutions could be made. The NNK library wasconstructed as described above using primer PEP-26-F to introducediversity at the selected positions in DOM23h-271-22 or DOM23h-271-102background. DOM23h-271-102 contains mutations at position 27 and 55 thatconfer improved affinity over DOM23h-271-7.

PEP-26-F  (SEQ ID NO: 209)GCGGTATATTACTGTGCGAAACAGNNSCCCGGCNNSAAGTGGNNSGCCNNSNNSCGCNNSGACTACTGGGGTCAGGGAACC

DNA display libraries were constructed and selected on biotinylatedhTGFbRII-FC as described above using concentrations of 5 nM; 0.5 nM; 0.1nM; 0.1 nM; 0.1 nM; and 0.1 nM in successive rounds. In selection rounds4-6 the non-biotinylated TGFbRII-FC competitor was added to a finalconcentration of 100 nM for 60, 90, and 90 minutes respectively beforeaddition of C1 streptavidin DYNAbeads™. Following selection the dAbinserts were PCR amplified using primers PeIB NcoVh and PEP011, cut withNcoI and EcoRI, and cloned into the bacterial expression vector pC10.

PelB NcoVh (SEQ ID NO: 210) GCCCAGCCGGCCATGGCGGAGGTGCAGCTGTTGGAGTCTGGGPEP011  (SEQ ID NO: 211) GAATTCGCGGCCGCCTATTAGCTCGAGACGGTGACCAGGG

The cloned products were expressed and screened by Biacore. Clones withoff-rates similar or better than DOM23h-271-22 were sequenced, purified,and assessed for affinity by BIACORE™ and potency in the cell sensorassay. Clones giving poor kinetic profiles, containing unfavourablesequence motifs, or giving very low yields were not pursued.DOM23h-271-50 was selected for further affinity maturation.

DOM-271-50 nucleic acid sequence  (SEQ ID NO: 212)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGGCGCCCGGCGAGAAGTGGCTCGCCCGGGGCCGCTTGGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM-271-50 amino acid sequence(SEQ ID NO: 213 and duplicate entry SEQ ID NO: 214)EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQAPGEKWLARGRLDYWGQGTLVTVSS

Example 7 Introduction of Mutations into TGFbRII dAb Sequences(DOM23h-271 Lineage) at Positions 61 and 64

The D61N and K64R double mutations have previously been introduced intovarious TGFRβII dAb lineages and have been shown to improve potency(WO2011/012609). These mutations were introduced into TGFbRII dAbDOM23h-271 lineages to see if a similar improvement in potency could beachieved. Alternative mutations at this position were explored todetermine whether further enhancements in potency could be achieved.

Mutations were introduced into the DOM23h-271 (SEQ ID NO:199) backboneby overlap extension using the polymerase chain reaction (PCR) (Ho, etal., Gene 1989 77(1)); complementary oligonucleotide primers were usedto generate two DNA fragments having overlapping or complementary ends.These fragments were combined in a subsequent assembly PCR in which theoverlapping ends anneal, allowing the 3′ overlap of each strand to serveas a primer for the 3′ extension of the complementary strand and theresulting fusion product to be amplified further by PCR. Specificalterations in the nucleotide sequence were introduced by incorporatingnucleotide changes into the overlapping oligonucleotide primers. Thetarget dAb gene fragments were amplified by two separate PCRs usingSUPERTAQ™ polymerase (HT Biotechnology). DOM23h-271 was selected as ithas a low affinity for TGFbRII so that improvements in affinity wereclearly measurable using BIACORE™. The mutations at positions 61 and 64were encoded using a degenerate oligo-nucleotide to mutate bothpositions in combination, with the intension of using affinity selectionto enrich for mutants with improved binding affinity. As the NR mutationis known to dominate this mutation was avoided in the library byemploying a restricted codon, NNG, at position 61. This codon encodes 13amino acids but does not include the amino acids F, Y, C, H, I, N or D.Free Cys was not favoured within a dAb, and asparagine was also notwanted at this position, so only 5 desirable amino acids combinationswere excluded. At position 64 an NNK codon was used to provide the fullrange of possible amino acids.

The oligonucleotide pairs used to introduce the mutations were PE008(flanks the 5′ start of the dAb gene:5′-TTGCAGGCGTGGCAACAGCGTCGACAGAGGTGCAGCTGTTGGAG-3′) (SEQ ID NO:192) with271-6164 R (5′-GCGTAGTATGTACGATTACCAATCGG-3′) (SEQ ID NO:193) andmutated 271-6164 deg-F (mutated residues underlined:5f-GGTAATCGTACATACTACGCANNGTCCGTGNNKGGCCGGTTCACCATCTCCCGC-3′) (SEQ IDNO: 194) with AS1309 (flanks the 3′ end of the dAb gene:5′-TGTGTGTGTGTGGCGGCCGCGCTCGAGACGGTGACCAGGGTTCCCTGACCCCA-3′) (SEQ IDNO:195). The two PCR fragments were recombined in an assembly PCR usingSUPERTAQ™ DNA polymerase without the addition of primers. The fusionproduct was then amplified by the addition of flanking primers PE008 andAS1309 to the PCR reaction. The mutated dAb was digested with Sal I andNot I restriction endonucleases, ligated into similarly cut pDOM13expression vector and transformed into E. Coli HB2151 cells. The D61N,K64R mutation was also introduced into DOM23h-271 in the same way, butsubstituting primer 271-6164 NR—F(5′-GGTAATCGTACATACTACGCAAACTCCGTGCGCGGCCGGTTCACCATCTCCCGC-3′) (SEQ IDNO:196) for 271-6164 deg-F. The mutated dAbs were identified bysequencing. 129 clones with novel combinations of mutations at position61 and 64 were identified, though 11 carried additional mutations fromPCR errors. These are shown in table 11. N.B. Clones with enhancedaffinity are underlined. Clones with additional mutations are indicatedby asterisks

To determine the effect of the mutations the selected clones wereexpressed in TB ONEX™ in 96 well plates for 72 hours at 30° C. or 24hours at 37° C. The supernatants were clarified by centrifugation andfiltered through a 0.22 μm filter before off-rate evaluation by BIACORE™

BIACORE™ A100 analysis was carried out using a capture surface on a CM5chip. Anti-human IgG was used as the capturing agent and coupled to aCM5 biosensor chip by primary amine coupling. The Antigen molecule fusedto the human Fc was captured on this immobilised surface to a level from200 to 300 resonance units and supernatants or purified domainantibodies were passed over this captured surface. An injection of mediaor buffer over the captured antigen surface was used for doublereferencing. On each flow cell, a protein A spot allowed to confirm thepresence of domain antibody on each sample injected. The surfaces wereregenerated, after each domain antibody injection using 3M magnesiumchloride solution; the regeneration removed the captured antigen but didnot significantly affect the ability of the surface to capture antigenin a subsequent cycle. All runs were carried out at 25° C. using HBS-EPbuffer as running buffer. Data were generated using the BIACORE™ A100and analyzed using its inherent software. Kinetics from purified sampleswere fitted to the 1:1 binding model while supernatant off-rates wereanalyzed using the 1:1 dissociation model and/or using the binding leveland the stability level of each sample in comparison to the parentmolecule.

Of clones tested 46 were identified with improved off-rate by comparisonto the parent DOM23h-271 dAb, as indicated in table 11. Mutations D61Rand D61K were found to enhance binding independent of mutations atposition 64. A number of combinations appeared better than the originalD61N, K64R mutations, and these included RE, RM, RF and RY.

A selection of mutants with improved off-rate were purified andsubjected to full BIACORE™ A100 kinetic analysis to determine theiraffinity (Table 10). The thermal stability of the mutants was alsodetermined by generation of melting profiles in the presence of SYPRO™Orange (Invitrogen). Briefly, the purified dAbs were diluted to 50 and100 ug/ml in a 1:500 dilution of SYPRO™ Orange in PBS, and subjected toa 30-90° C. melt curve in a Mini OPTICON™ PCR machine (BioRad). The Tmof the major positive transition was determined at each concentrationand used to calculate an estimated Tm value at 1 mg/ml as an indicationof the melting temperature for comparison (Table 10).

The mutations D61R, K64D and D61R, K64F were selected as they had a goodimprovement in affinity for a reduced impact in Tm. These mutations weretransferred to an affinity matured DOM23h-271 derivative DOM23h-271-22(SEQ ID NO:30) to make dAbs DOM23h-271-39 (SEQ ID NO:37) andDOM23h-271-40 (SEQ ID NO:38). These mutations were found to give anenhancement in affinity by BIACORE™ including cross reactivity withmurine TGFbRII FC, and enhanced potency by cell sensor assay. Howeverthe mutations were also found to reduce the Tm as measured by DSC, andincrease aggregation of the dabs in PBS, as measured by SEC MALLS. Theseassays were carried out as described above, except that the buffer forSEC MALLS was 0.4M Nacl, 0.1M Sodium Phosphate and 10% isopropanol pH7.These results are summarized in table 12 (mean values are given for thehuman cell sensor assay and the mouse 3T3 luciferase assay).

Sequences of dabs referred to in this example are given below:

DOM23h-271-39 amino acid sequence (SEQ ID NO: 37)EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYARSVDGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQMPGQKWMAKSRFDYWGQGTLVTVSSDOM23h-271-39 nucleic acid sequence  (SEQ ID NO: 75)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTAATCGTACATACTACGCACGCTCCGTGGACGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGATGCCCGGCCAGAAGTGGATGGCCAAGTCCCGCTTCGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCDOM23h-271-40 amino acid sequence  (SEQ ID NO: 38)EVQLLESGGGLVQPGGSLRLSCAASGFTFTEYRMWWVRQAPGKGLEWVSAIEPIGNRTYYARSVFGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQMPGQKWMAKSRFDYWGQGTLVIVSSDOM23h-271-40 nucleic acid sequence  (SEQ ID NO: 76)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTAATCGTACATACTACGCACGCTCCGTGTTCGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGATGCCCGGCCAGAAGTGGATGGCCAAGTCCCGCTTCGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC

TABLE 10 Description Mutation Est. Tm 1 mg/ml KD (nM) 1A2 RA 53.08 2.241D9 RD 52.35 1.17 3F11 RE 53.08 3.27 1G11 RM 51.81 0.97 3C9 RF 54.081.39 1E6 RY 49.62 1.17 1B11 RV 53.41 1.88 3D9 KG 48.69 4.34 1D5 KF 48.093.69 3D4 KT 54.74 4.26 1H11 LW 53.68 4.17 1B2 VW 52.68 1.79 2C12 NR51.49 3.34 271 DK 58.21 N/D N.B. the mutation corresponds to ‘XY’wherein X is position 61 and Y is position 64

TABLE 11 Mutations inserted into DOM23h-271 at positions 61 and 64.61\64 A R N D C Q E G H I L K M A 1D6* 1C10 3B11 R 1A2 1A3 3F4 1D9 2C41A8 3F11 3G8 1A4 3C2 1G11 N 2C12 D 3H6 (wt) C Q 1B6* 1E8 1B10 1H12 1D11E 2A3 1F12 2G4 3H7 G 1D1 1B5 3B12 1E5 3G11 3D12 H I L 1D2 3E11 2A9 1B41H3 1A9 1F1 1G9 1H10 1C9 K 2E5 — 2B4 3D9 3E12 M 1G6 2D3* 1E7 2E6 3C12 FP 2A8 1A5 2C11 S 3C3 3E7 3E5 1H4 2B10 1C11 T 1E3 1D10* 2C1 1C1 2A5 3G9 W2F10 1E12 2C2 2A2 2A6 1D4 Y V 1C12 2B9 1H6 1D3* 3C1 2G2 2F7 1F7 61\64 FP S T W Y V A 3C10* 3B10 1A12 3G3* R 3C9* 2F5 1H1 1H2 1E6 1B11 N D C Q1C8 3F7 2C10 E 1F10 2B11 3D6 G 2B6 2G5 2D5 H I L 2C9 1H5 1G2 1E4 1H11 K1D5 1G7 2E9* 3D4 1B1 1F2 M 2F6 3H1 3B5* 1A6 1E11 3B8* F P 3H2 1G5 3G12 S1B7 2C8 3B1 3D5 T 2H10 2A4 3D11 W 3B2 2B1 Y V 2H4 3C7 1B2 2G6 Cloneswith improved off-rate are underlined. Clones with additional mutationsare indicated by asterisks

TABLE 12 Human Cell Mouse 3T3 Human Cyno Mouse sensor Luciferase assayTGFbRII TGFbRII TGRbRII Tm Aggregation Mutation IC50 (nM) IC50 (nM) KD(pM) KD (pM) KD (pM) (DSC) (SEC-MALS) DOM23h-271- — 39.01 >17780 202.00225.00 — 60 91.8% Monomer 22  8.2% Dimer DOM23h-271- RD  1.17   11120 14.90  24.00 1780.00 57.5 51.6% M/D Eq 39 38.5% D/T Eq + aggregatesDOM23h-271- RF  0.53    1200  9.99  11.25  548.50 55.6 91.6% M/D Eq 40 8.4% Trimer/ oligomer

Example 8 Affinity Maturation by CDR Diversification of LineagesDOM23h-439-20, DOM23h-843-13, DOM23h-855-21 and DOM23h-271-50

Domain antibodies DOM23h-855-21(SEQ ID NO:204) and DOM23h-843-13 (SEQ IDNO:206) were isolated from the error prone affinity maturation carriedout on naive clones DOM23h-855 (SEQ ID NO:13) and DOM23h-843 (SEQ IDNO:10) respectively, as detailed in example 5. Domain antibodyDOM23h-439 was isolated from phage libraries in a previous selectioncampaign described in WO 2011/012609 and variant DOM23h-439-20 (SEQ IDNO:208) was subsequently isolated by error prone affinity maturation asdetailed in example 5. Domain antibody DOM23h-271-50 (SEQ ID NO:214) wasgenerated by CDR3 re-diversification of CDR-directed affinity maturedvariant DOM23h-271-22 (SEQ ID NO:30) as detailed in Example 6.DOM23h-855-21, DOM23h-843-13, DOM23h-439-20 and DOM23h-271-50 were allselected for further affinity maturation based on their bindingkinetics, potency in the SBE-bla HEK 293T cell sensor assay, sequenceand biophysical behaviour. Affinity maturation was performed usingdegenerative mutagenesis to re-diversify the CDRs, and improved leadswere identified using phage display. Diversity was introduced into theCDRs by construction of either doped or NNK libraries.

DOM23h-271-50 was affinity matured using NNK libraries (saturationmutagenesis), oligonucleotide primers were designed to cover each CDRand within each primer the codons for 5 amino acids were replaced withNNK codons so that 5 positions are diversified simultaneously. Single ormultiple oligonucleotides were used to cover all targeted amino acidswithin each CDR; 1 for CDR1, 2 for CDR2 and 3 for CDR3. CDR-directedaffinity maturation was achieved using polymerase chain reaction (PCR);Complementary oligonucleotide primers were used to amplify a sequencefragment containing each mutated CDR, and also an overlapping sequencefragment covering the rest of the dAb coding sequence. These fragmentswere mixed and assembled by splice extension overlap PCR to produce thefull length dAb coding sequence. This product was PCR amplified usingprimers PeIB NcoVh (SEQ ID NO:210) and PEP044(5′-GGAACCCTGGTCACCGTCTCGAGCGCGGCCGCATAATAAGAATTCA-3′ SEQ ID NO:215),digested with NcoI and NotI, and ligated into NotI and NcoI digestedpDOM4-gene3-pelB hybrid vector. pDOM4-gene3-pelB hybrid vector is amodified version of the pDOM4 vector described above but has beenmodified to replace the GAS leader sequence with the pelB (pectate lyaseB) signal peptide.

The domain antibodies DOM23h-855-21, DOM23h-843-13 and DOM23h-439-20were affinity matured using doped libraries. The doped libraries wereconstructed essentially as described above and in WO2006018650. A singledegenerate oligonucleotide primer was used to cover all mutations withineach CDR. Within each primer the amino acids to be diversified werespecified using degenerate codons to encode multiple amino acids. Thefollowing degenerate coding was used: ‘a’=85% A+5% T+5% G+5% C; ‘g’=85%G+5% T+5% C+5% A; ‘c’=85% C+5% T+5% G+5% A; ‘t’=85% T+5% A+5% G+5% C.;‘S’=50% G and 50% C. Capital letters indicate 100% of the specifiednucleotide. For the DOM23h-439-20 doped library five amino acids werediversified in CDR1, 6 in CDR2 and 11 in CDR3 to include thephenyalanine at position 100. For the DOM23h-855-21 doped library fiveamino acids were diversified in CDR1, 7 in CDR2 and 8 in CDR3. For theDOM23h-843-13 doped library five amino acids were diversified in CDR1, 6in CDR2 and 8 in CDR3, position 94 before the CDR3 was also diversifiedby introducing the codon VRK. For each of the dAbs, 4 doped librarieswere constructed, one to diversify CDR1, one to diversify CDR2, one todiversify CDR3 and a fourth where all CDRs were diversified. Thedegenerate library primers were used in the same way as the tripletprimers. Each diversified CDR was amplified separately and then combinedwith a parental sequence fragment using splice extension overlap, thefull length product was amplified using primers PeIB NcoVh (SEQ IDNO:210) and DOM173 (SEQ ID NO:188). The fragments were subcloned topDOM4-gene3-pelB hybrid vector using NcoI and NotI.

Degenerate Primer Sequences:

23h-439-20 CDRH1  (SEQ ID NO: 216)5′-GCAGCCTCCGGATTCACCTTTggSacSgagcagATGtggTGGGTCCGCCAGGCTCCAGGG-3′23h-439-20 CDRH2  (SEQ ID NO: 217)5′-AAGGGTCTAGAGTTTGTCTCAcgSATTgattcSccSGGTggScgSACATACTACGCAGACTCCGTG-3′23h-439-20 CDRH3  (SEQ ID NO: 218)5′-GCGGTATATTACTGTGCGAAAcgScatgcSgcSggSgtStcSggSacStaYtttGACTACTGGGGTCAGGGAACC-3′23h-843-13 CDRH1  (SEQ ID NO: 219)5′-GCAGCCTCCGGATTCACCTTTgatcaggatcgSATGtggTGGGTCCGCCAGGCCCCAGGG-3′23h-843-13 CDRH2  (SEQ ID NO: 220)5′-AAGGGTCTAGAGTGGGTCTCAgcSATTgagtcSggSGGTcatcgSACATACTACGCAGACTCCGTG-3′23h-843-13 CDRH3  (SEQ ID NO: 221)5′-ACCGCGGTATATTACTGTGCGVRKcagaataagtcSggScgStcSggSTTTGACTACTGGGGTCAGGGA-3′23h-855-21 CDRH1  (SEQ ID NO: 222)5′-GCAGCCTCCGGATTCACCTTTgagaatacStcSATGggSTGGGTCCGCCAGGCTCCAGGG-3′23h-855-21 CDRH2  (SEQ ID NO: 223)5′-AAGGGTCTAGAGTGGGTCTCAcgSATTgatccSaagGGTtcScatACATACTACacSGACTCCGTGAAGGGCCGGTTCACC-3′ 23h-855-21 CDRH3  (SEQ ID NO: 224)5′-GCGGTATATTACTGTGCGAAAcagcgSgagctSggSaagtcStaYTTTGACTACTGGGGTCAGGGA-3′H1-271-43 R  (SEQ ID NO: 225)5′-GCAGCCTCCGGATTCACCTTTNNKNNKNNKNNKATGNNKTGGGTCCGCCAGGCTCCGGGGAAGGGTCTC-3′H2p1-271-43F  (SEQ ID NO: 226)5′-CCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCANNKATTNNKNNKNNKGGTNNKCGTACATACTACGCAGACTCCG-3′ H2p2-271-43 F  (SEQ ID NO: 227)5′-CCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGNNKNNKNNKNNKNNKACATACTACGCAGACTCCG-3′ H3p1-271-43 F  (SEQ ID NO: 228)5′-ACCGCGGTATATTACTGTGCGAAANNKNNKNNKNNKNNKAAGTGGATGGCCGTGGGCCGCTTGGACTACTGGGGTCAGGG-3′ H3p2-271-43 F  (SEQ ID NO: 229)5′-ACCGCGGTATATTACTGTGCGAAACAGAAGCCCNNKNNKNNKNNKNNKGCCGTGGGCCGCTTGGACTACTGGGGTCAGGG-3′ H3p3-271-43 F  (SEQ ID NO: 230)5′-ACCGCGGTATATTACTGTGCGAAACAGAAGCCCGGCCAGAAGTGGNNKNNKNNKNNKNNKTTGGACTACTGGGGTCAGGG-3′Phage Display:

NNK or doped libraries in separate CDR1, CDR2, CDR3 and combined CDR1, 2and 3 pools were subjected to at least 6 rounds of selection against 100nM, 10 nM, 1 nM, 1 nM, 0.1 nM and 0.1 nM (rounds 1, 2, 3, 4, 5 and 6respectively) biotinylated monomeric human TGF-β RII antigen asdescribed in example 1 with the following deviation to the block step;the human IgG Fc fragment which was previously added in example 1 wasomitted and the block step performed for a minimum of 20 minutes. Inrounds 4 and 6 of selection competition was introduced by incubationwith 100 nM non-biotinylated antigen for 30 min (rounds 4 and 6)following the incubation step with the labelled antigen. For DOM23h-439-20 (CDR1, CDR3 and combined pools) and DOM 23h-855-21 (CDR3) aseventh round of selection was carried out against 0.1 nM biotinylatedmonomeric human TGF-β RII antigen and 100 nM competition for 120 min or20 pM biotinylated monomeric human TGF-β RII antigen with 100 nMcompetition for 30 min. Phage were amplified between rounds of selectionby centrifugation of an overnight culture of phage infected TG1 cellsfor 30 minutes at 4000 g. 40 ml of supernatant containing the amplifiedphage was added to 10 ml of PEG/NaCl (20% v/w PEG 8000+2.5 M NaCl) andincubated on ice for 60 minutes. The samples were centrifuged for 40minutes at 4000 g to pellet the precipitated phage. The supernatant wasdiscarded and the phage pellet was resuspended in 1 ml 15% v/vglycerol/PBS. The phage sample was transferred to 2 ml Eppendorf tubesand centrifuged for 10 minutes to remove any remaining bacterial celldebris. Diversified domain antibody Vh genes from the phage selectionswere PCR amplified using primers PEP011VHStopNotIR(5′-CCCTGGTCACCGTCTCGAGCTAATAGGCGGCCGCGAATTC-3′ (SEQ ID NO: 231) andNco1 VH F (5′-TATCGTCCATGGCGGAGGTGCAGCTGTTGGAGTCTGG-3′ (SEQ ID NO: 232),digested with NcoI and NotI restriction endonucleases and ligated intothe pC10 vector also digested with NcoI and NotI. pC10 is a plasmidvector based on pUC119 vector, with expression under the control anenhanced LacZ promoter designed for soluble expression of dAbs.Expression of dAbs into the supernatant is enabled by fusion of the dAbgene to the pelB signal peptide at the N-terminal end. The ligationproducts were transformed into chemically competent E. coli HB2151 cellsand plated on nutrient agar plates supplemented with 100 μg/ml ofcarbenicillin. Individual clones were picked and expressed in overnightexpress auto-induction medium (high-level protein expression system,Novagen), supplemented with 100 μg/ml carbenicillin in 96-well platesand grown with shaking at 250 rpm for either 66 hours at 30° C. or 24hours at 37° C. Expression plates were then centrifuged at 3500 g for 15minutes and the supernatants filtered using 0.45μ filter plates(Millipore). Supernatants containing the domain antibodies were screenedon the BIACORE™ B4000 against human TGF-β RII and human TGF-β RII/Fc todetermine the off-rate (Kd) (data not shown). Biotinylated antigens werecaptured on an SA chip in accordance with the manufacturer'sinstructions, analysis was carried out at 25° C. using HBS-EP buffer.Samples were run on BIACORE™ at a flow rate of 30 μl/min. Regenerationof the chip was achieved using glycine at low pH. The data (not shown)were analysed for off-rate by fitting the dissociation phase to the 1:1dissociation model inherent to the software, supernatants were alsoanalyzed for protein A binding to estimate levels of expression. Domainantibodies with off rates improved over parent were selected for furtherstudy. Improved clones were expressed in overnight express autoinductionmedium (ONEX™, Novagen) supplemented with 100 ug/ml carbenicillin andantifoam (Sigma) at 30° C. for 48 to 72 hours with shaking at 250 rpm.The cultures were centrifuged (4,200 rpm for 40 minutes) and thesupernatants were incubated with STREAMLINE™-protein A beads (AmershamBiosciences, GE HEALTHCARE™, UK. Binding capacity: 5 mg of dAb per ml ofbeads), at 4° C. or at room temperature for at least one hour. The beadswere packed into a chromatography column and washed with PBS, followedby 10 mM Tris-HCl pH 7.4 (Sigma, UK). Bound dAbs were eluted with 0.1 Mglycine-HCl pH 2.0 and neutralized with 1M Tris-HCL pH 8.0. The OD at280 nm of the dAbs was measured and protein concentrations weredetermined using extinction coefficients calculated from the amino acidcompositions of the dAbs. Affinity matured domain antibodies were testedon the BIACORE™ T200 (data for two preferred dAbs are shown in Example9) and in the SBE-bla HEK 293T Cell Sensor assay (data for two preferreddAbs are shown in Example 11) to determine their affinity and potency.Biophysical properties including thermal stability and solution statewere determined using Differential Scanning Colourimetry (DSC) and sizeexclusion chromatography with multi-angle-LASER-light scattering(SEC-MALS) (data for two preferred dAbs are shown in Example 10).

The amino acid and nucleic acid sequences of affinity maturedDOM23h-439-20 and DOM23h-271-50 anti-human TGFRII dAbs

DOM23h-439-25 nucleic acid sequence  (SEQ ID NO: 233)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTGTCTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACGGCGACCCACGGGGGTGTCCGGGACGATGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23h-439-25 amino acid sequence  (SEQ ID NO: 234)EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFVSRIDSPGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRPTGVSGTFYDYWGQGTLVTVSSDOM23h-271-123 nucleic acid sequence  (SEQ ID NO: 235)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGGCGCCCGGCGAGAAGTGGGCGAGGCGGTGGGATTTGGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23h-271-123 amino acid sequence  (SEQ ID NO: 236)EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQAPGEKWARRWDLDYWGQGTLVTVSSDOM23h-439-35 nucleic acid sequence  (SEQ ID NO: 237)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGGGACCGATCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTGTCTCACGCATTGATTCCCCCGGTGGGCGGACATACTACGCAAACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACGGCAGCCGGCGGGGGTGTCGGGGAAGTACGTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTC TCGAGCDOM23h-439-35 amino acid sequence  (SEQ ID NO: 238)EVQLLESGGGLVQPGGSLRLSCAASGFTFGTDQMWWVRQAPGKGLEFVSRIDSPGGRTYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRQPAGVSGKYVDYWGQGTLVTVSSDOM23h-271-129 nucleic acid sequence  (SEQ ID NO: 239)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGGCGCCCAATCAGAGGTATGTTGCCCGGGGCCGCTTGGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC DOM23h-271-129 amino acid sequence  (SEQ ID NO: 240)EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQAPNQRYVARGRLDYWGQGTLVIVSS

The CDRs as defined by Kabat of these anti-human TGFRII dAb affinityleads are shown in Table 13

TABLE 13 CDR Sequences of affinity matured anti-human TGFβRII dAbsCDR1 (Kabat 26-35) CDR2 (Kabat 50-65) CDR3 (Kabat 95-102) DOM23h-GSTFTEYRMW AIEPIGHRTYYADSVKG QAPGEKWARRWDLDY 271423 (SEQ ID NO: 241)(SEQ ID NO: 242) (SEQ ID NO: 243) DOM23h- GSTFTEYRMW AIEPIGHRTYYADSVKGQAPNQRYVARGRLDY 271429 (SEQ ID NO: 244) (SEQ ID NO: 245)(SEQ ID NO: 246) DOM23h- GFTFGTEQMW RIDSPGGRTYYADSVKG RRPTGVSGTFYDY439-25 (SEQ ID NO: 247) (SEQ ID NO: 248) (SEQ ID NO: 249) DOM23h-GFTFGTDQMW RIDSPGGRTYYANSVKG RQPAGVSGKYVDY 439-35 (SEQ ID NO: 250)(SEQ ID NO: 251) (SEQ ID NO: 252)Further Modification of the DOM23h-439-25 and DOM23h-271-123 NucleotideSequence

The D61N and K64R mutations as described in example 7 were introducedinto the DOM23h-439-25, DOM23h-271-123 and DOM23h-271-129 affinitymatured dAbs either in combination or separately. Introduction of a V48Imutation in the DOM23h-439-25 and DOM23h-271-123 dAbs was also tested todetermine whether it could confer improvements in potency. Spontaneousmutation at kabat position 48 was observed in a number of improved dAbsfrom the DOM23h-439 lineage following both test maturation andCDR-directed affinity maturation. An Alanine at the C-terminus of the Vhregion, immediately after kabat residue 113 was also added toDOM23h-439-25, DOM23h-271-123 and DOM23h-271-129 and all variants ofthese dAbs with the afore-mentioned mutations. Mutations were introducedinto the DOM23h-439-25 (SEQ ID NO: 234), DOM23h-271-123 (SEQ ID NO: 236)and DOM23h-271-129 (SEQ ID NO: 240) backbones by overlap extension usingthe polymerase chain reaction (PCR) essentially as described in Example7. Specific mutations in the nucleotide sequence were introduced byincorporating nucleotide changes into the overlapping oligonucleotideprimers, the insertion of the Alanine at the end of the Vh region wasachieved by using a 3′ oligonucleotide designed to incorporate theAlanine residue after kabat position 113.

The following oligonucleotides were used to introduce the mutations(mutated residues underlined):

439 48I SDM F  (SEQ ID NO: 253) 5′-GGGTCTAGAGTTTATTTCACGTATTGATTCGCC-3′439 61N SDM F  (SEQ ID NO: 254)5′-GGGAGGACATACTACGCAAACTCCGTGAAGGGCCGG-3′ 439 64R SDM F (SEQ ID NO: 255) 5′-CGCAGACTCCGTGCGTGGCCGGTTCACC-3′ 439 61N 64R SDM F (SEQ ID NO: 256) 5′-GGGAGGACATACTACGCAAACTCCGTGCGTGGCCGGTTCACC-3′271 61N SDM F  (SEQ ID NO: 257) 5′-GGACATACTACGCAAACTCCGTGAAGGGCCGG-3′271 64R SDM F  (SEQ ID NO: 258) 5′-CGCAGACTCCGTGCGTGGCCGGTTCACC-3′271 61N 64R SDM F  (SEQ ID NO: 259)5′-GGACATACTACGCAAACTCCGTGCGTGGCCGGTTCACC-3′ 567 +A rev (Flanks the 3′end of the dAb Vh gene) (SEQ ID NO: 260)5′-CCCTGGTCACCGTCTCGAGCGCGTAATAAGCGGCCGCAGATTA-3′21-23 Fwd (Flanks the 5′ end of the dAb Vh gene) (SEQ ID NO: 261)5′-ATAAGGCCATGGCGGAGGTGCAGCTGTTGGAGTCTG-3′

To determine the effect of the mutations the dAbs were expressed in TBONEX™ supplemented with 100 ug/ml carbenicillin and antifoam (Sigma) at30° C. for 72 hours with shaking at 250 rpm. The cultures werecentrifuged (4,200 rpm for 40 minutes) and dAbs affinity purified usingSTREAMLINE™-protein A beads (Amersham Biosciences, GE HEALTHCARE™, UK)as before. Affinity and potency of the domain antibody variants weredetermined on the BIACORE™ T200 (data for preferred dAbs is shown inExample 9) and in the SBE-bla HEK 293T Cell Sensor assay (data forpreferred dAbs is shown in Example 11).

The amino acid and nucleic acid sequences of DOM23h-439-25 (SEQ ID NO:234) and DOM23h-271-123 (SEQ ID NO: 236) anti-human TGFRII dAbs modifiedto include the C-terminal Alanine and D61N, K64R or V48I mutations aregiven below:

DOM23h-439-40 (DOM23h-439-25 + C-terminal Alanine) Nucleic acid sequence(SEQ ID NO: 262)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTGTCTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACGGCGACCCACGGGGGTGTCCGGGACGATGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCG DOM23h-439-40 (DOM23h-439-25 +C-terminal Alanine) Amino acid sequence  (SEQ ID NO: 263)EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFVSRIDSPGGRMADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRPTGVSGTFYDYWGQGTLVTVSSADOM23h-439-41 (DOM23h-439-25 + C-terminal Alanine +48I) Nucleic acid sequence  (SEQ ID NO: 264)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTATTTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACGGCGACCCACGGGGGTGTCCGGGACGATGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCG DOM23h-439-41 (DOM23h-439-25 + C-terminal Alanine +48I) Amino acid sequence  (SEQ ID NO: 265)EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFISRIDSPGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRPTGVSGTFYDYWGQGTLVTVSSADOM23h-439-42 (DOM23h-439-25 + C-terminal Alanine +61N) Nucleic acid sequence  (SEQ ID NO: 266)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTGTCTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAAACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACGGCGACCCACGGGGGTGTCCGGGACGATGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCG DOM23h-439-42 (DOM23h-439-25 + C-terminal Alanine +61N) Amino acid sequence (SEQ ID NO: 267)EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFVSRIDSPGGRTYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRPTGVSGTFYDYWGQGTLVTVSSADOM23h-439-43 (DOM23h-439-25 + C-terminal Alanine +64R) Nucleic acid sequence (SEQ ID NO: 268)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTGTCTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAGACTCCGTGCGTGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACGGCGACCCACGGGGGTGTCCGGGACGATGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCG DOM23h-439-43 (DOM23h-439-25 + C-terminal Alanine +64R) Amino acid sequence (SEQ ID NO: 269)EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFVSRIDSPGGRTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRPTGVSGTFYDYWGQGTLVTVSSADOM23h-439-44 (DOM23h-439-25 + C-terminal Alanine +61N64R) Nucleic acid sequence (SEQ ID NO: 270)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTGTCTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAAACTCCGTGCGTGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACGGCGACCCACGGGGGTGTCCGGGACGATGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCG DOM23h-439-44 (DOM23h-439-25 + C-terminal Alanine +61N64R) Amino acid sequence (SEQ ID NO: 271)EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFVSRIDSPGGRTYYANSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRPTGVSGTFYDYWGQGTLVTVSSADOM23h-271-130 (DOM23h-271-123 +C-terminal Alanine) Nucleic acid sequence (SEQ ID NO: 272)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGGCGCCCGGCGAGAAGTGGGCGAGGCGGTGGGATTTGGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCG DOM23h-271-130 (DOM23h-271-123 +C-terminal Alanine) Amino acid sequence (SEQ ID NO: 273)EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQAPGEKWARRWDLDYWGQGTLVTVSSADOM23h-271-131 (DOM23h-271-123 + C-terminal Alanine +61N) Nucleic acid sequence (SEQ ID NO: 274)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAAACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGGCGCCCGGCGAGAAGTGGGCGAGGCGGTGGGATTTGGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCG DOM23h-271-131 (DOM23h-271-123 +C-terminal Alanine + 61N) Amino acid sequence (SEQ ID NO: 275)EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQAPGEKWARRWDLDYWGQGTLVTVSSADOM23h-271-132 (DOM23h-271-123 + C-terminal Alanine +64R) Nucleic acid sequence (SEQ ID NO: 276)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAGACTCCGTGCGTGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGGCGCCCGGCGAGAAGTGGGCGAGGCGGTGGGATTTGGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCG DOM23h-271-132 (DOM23h-271-123 +C-terminal Alanine + 64R) Amino acid sequence (SEQ ID NO: 277)EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQAPGEKWARRWDLDYWGQGTLVTVSSADOM23h-271-133 (DOM23h-271-123 + C-terminal Alanine +61N64R) Nucleic acid sequence (SEQ ID NO: 278)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAAACTCCGTGCGTGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGGCGCCCGGCGAGAAGTGGGCGAGGCGGTGGGATTTGGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCG DOM23h-271-133 (DOM23h-271-123 +C-terminal Alanine + 61N64R) Amino acid sequence (SEQ ID NO: 279)EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYANSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQAPGEKWARRWDLDYWGQGTLVTVSSADOM23h-271-134 (DOM23h-271-123 + C-terminal Alanine +48I) Nucleic acid sequence (SEQ ID NO: 280)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGATTTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGGCGCCCGGCGAGAAGTGGGCGAGGCGGTGGGATTTGGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCG DOM23h-271-134 (DOM23h-271-123 +C-terminal Alanine + 48I) Amino acid sequence (SEQ ID NO: 281)EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWISAIEPIGHRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQAPGEKWARRWDLDYWGQGTLVTVSSADOM23h-271-135 (DOM23h-271-129 +C-terminal Alanine) Nucleic acid sequence (SEQ ID NO: 282)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGGCGCCCAATCAGAGGTATGTTGCCCGGGGCCGCTTGGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCG DOM23h-271-135 (DOM23h-271-129 +C-terminal Alanine) Amino acid sequence (SEQ ID NO: 283)EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQAPNQRYVARGRLDYWGQGTLVTVSSADOM23h-271-136 (DOM23h-271-129 + C-terminal Alanine +61N) Nucleic acid sequence (SEQ ID NO: 284)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAAACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGGCGCCCAATCAGAGGTATGTTGCCCGGGGCCGCTTGGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCG DOM23h-271-136 (DOM23h-271-129 + C-terminal Alanine +61N) Amino acid sequence (SEQ ID NO: 285)EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQAPNQRYVARGRLDYWGQGTLVTVSSADOM23h-271-137 (DOM23h-271-129 + C-terminal Alanine +61N64R) Nucleic acid sequence (SEQ ID NO: 286)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATCCACCTTTACGGAGTATAGGATGTGGTGGGTCCGCCAGGCTCCGGGGAAGGGTCTCGAGTGGGTCTCAGCGATTGAGCCGATTGGTCATAGGACATACTACGCAAACTCCGTGCGTGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGATACCGCGGTATATTACTGTGCGAAACAGGCGCCCAATCAGAGGTATGTTGCCCGGGGCCGCTTGGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCG DOM23h-271-137 (DOM23h-271-129 + C-terminal Alanine +61N64R) Amino acid sequence (SEQ ID NO: 287)EVQLLESGGGLVQPGGSLRLSCAASGSTFTEYRMWWVRQAPGKGLEWVSAIEPIGHRTYYANSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQAPNQRYVARGRLDYWGQGTLVTVSSADOM23h-439-47 (DOM23h-439-42 + 48I) Nucleic acid sequence (SEQ ID NO: 288)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTATTTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAAACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACGGCGACCCACGGGGGTGTCCGGGACGATGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCG DOM23h-439-47 (DOM23h-439-42 + 48I) Amino acid sequence (SEQ ID NO: 289)EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFISRIDSPGGRTYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRPTGVSGTFYDYWGQGTLVTVSSADOM23h-439-48 (DOM23h-439-44 + 48I) Nucleic acid sequence (SEQ ID NO: 290)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGGGACGGAGCAGATGTGGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTTTATTTCACGTATTGATTCGCCTGGTGGGAGGACATACTACGCAAACTCCGTGCGTGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAACGGCGACCCACGGGGGTGTCCGGGACGATGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCG DOM23h-439-48 (DOM23h-439-44 + 48I) Amino acid sequence (SEQ ID NO: 291)EVQLLESGGGLVQPGGSLRLSCAASGFTFGTEQMWWVRQAPGKGLEFISRIDSPGGRTYYANSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRPTGVSGTFYDYWGQGTLVTVSSA

Example 9 Biacore Kinetic Analysis of Affinity Matured Domain Antibodies

Anti-human IgG was immobilised on a Biacore CM4 chip by primary aminecoupling according to the manufacturer's instructions. Human TGF-βRII/Fc, cynomolgus TGF-β RII/Fc or human Fc fragment were captured onthis surface. Domain antibodies were passed over the two capturedreceptors at 3 concentrations of 100, 10 and 1 nM (DOM23h-439 dAbs) or100, 25 and 6.25 nM (DOM23h-271 dAbs). Only the 100 nM concentration ofeach dab was passed over human Fc fragment to confirm specificity ofbinding to the extracellular TGF-β RII domain. An injection of bufferover the captured antigen surface was used for double referencing. Thecaptured surface was regenerated, after each domain antibody injectionusing 3M magnesium chloride solution; the regeneration removed thecaptured antigen but did not significantly affect the ability of thesurface to capture antigen in a subsequent cycle. All runs were carriedout at 25° C. using HBS-EP buffer as running buffer. Data were generatedusing the BIACORE™ T200 and fitted to the 1:1 binding model inherent tothe software. Table 14 shows the binding kinetics of the dAbs tested.The DOM23h-271 dAbs and the DOM23h-439 lineages were run in separateexperiments.

TABLE 14 Human TGFBRII Cyno TGFBRII sample Ka (M⁻¹ · s⁻¹) Kd (s⁻¹) KD(M) Ka (M⁻¹ · s⁻¹) Kd (s⁻¹) KD (M) DOM23h-271-123 2.89E+06 2.93E−041.02E−10 3.08E+06 3.20E−04 1.04E−10 DOM23h-271-129 5.55E+06 4.11E−047.41E−11 6.00E+06 4.27E−04 7.12E−11 DOM23h-271-130 2.51E+06 3.09E−041.23E−10 2.61E+06 3.24E−04 1.24E−10 DOM23h-271-131 6.26E+06 1.36E−042.17E−11 6.81E+06 1.44E−04 2.12E−11 DOM23h-271-132 1.22E+07 2.22E−041.82E−11 1.29E+07 2.38E−04 1.85E−11 DOM23h-271-133 8.47E+06 7.70E−059.09E−12 8.84E+06 8.71E−05 9.85E−12 DOM23h-439-25  7.77E+06 6.34E−048.17E−11 8.99E+06 2.73E−03 3.04E−10 DOM23h-439-35  2.26E+07 2.22E−049.83E−12 2.32E+07 6.75E−04 2.91E−11 DOM23h-439-40  1.34E+07 1.39E−031.04E−10 9.34E+06 3.42E−03 3.66E−10 DOM23h-439-41  1.81E+07 1.87E−041.04E−11 1.57E+07 5.22E−04 3.33E−11 DOM23h-439-42  4.09E+07 1.31E−043.20E−12 3.72E+07 4.10E−04 1.10E−11 DOM23h-439-43  8.83E+06 3.73E−044.23E−11 8.04E+06 1.20E−03 1.49E−10 DOM23h-439-44  2.39E+07 6.91E−052.89E−12 2.17E+07 1.47E−04 6.77E−12

Example 10 Biophysical Evaluation of Affinity Matured dAbs

The thermal stability of the dAbs was determined using DifferentialScanning calorimeter (DSC). dAbs were dialysed overnight into PBS bufferand adjusted at a final concentration of 1 mg/ml. Dialysis buffer wasused as a reference for all samples. DSC was performed using capillarycell microcalorimeter VP-DSC (GE healthcare/Microcal), at a heating rateof 180° C./hour. A typical scan range was from 20-90° C. for both thereference buffer and the protein sample. After each reference buffer andsample pair, the capillary cell was cleaned with a solution of 5% Decon(Fisher-Scientific) in water followed by PBS. Resulting data traces wereanalyzed using Origin 7.0 software. The DSC trace obtained from thereference buffer was subtracted from the sample trace. The precise molarconcentration of the sample was entered into the data analysis routineto yield values for melting temperature (Tm), enthalpy (ΔH) and VaraHoff enthalpy (ΔHv) values. Data were fitted to a non-2-state model.Best fit and dependencie values were obtained with either 1 or 2transition events. On-set unfolding temperature was also determined byintegrating from zero each sample thermogram. This value was thendetermined as the temperature at which 4 percentage of the sample wasunfolded.

TABLE 14A On-set Protein name Apparent Tm (C.) temperature (C.)DOM23h-439-21 60.66 52.5 DOM23h-439-25 56.34 49 DOM23h-439-30 57.17 50DOM23h-439-32 55.01 49 DOM23h-439-33 58.93 51 DOM23h-439-34 56.78 50DOM23h-855-42 61.90 55 DOM23h-855-43 66.81 58.6 DOM23h-855-44 63.38 56DOM23h-271-123 54.82 50.6 DOM23h-271-124 58.16 52 DOM23h-271-125 58.2655Analysis of Solution State by Size Exclusion Chromatography withMulti-Angle-LASER-Light Scattering (SEC-MALS)

To determine whether dAbs are monomeric or form higher order oligomersin solution, they were analyzed by SEC-MALLS (Size ExclusionChromatography with Multi-Angle-LASER-Light-Scattering). Agilent 1100series HPLC system with an autosampler and a UV detector (controlled byEmpower software) was connected to Wyatt Mini Dawn Treos (Laser LightScattering (LS) detector) and Wyatt Optilab rEX DRI (DifferentialRefractive Index (RI) detector). The detectors are connected in thefollowing order -UV-LS-RI. Both RI and LS instruments operate at awavelength of 658 nm; the UV signal is monitored at 280 nm and 220 nm.Domain antibodies (50 microliters injection at a concentration of 1mg/mL in PBS) were separated according to their hydrodynamic propertiesby size exclusion chromatography using a TSK2000 column. The mobilephase was 0.2M NaCl, 0.1M NaPO4, 15% n-propanol. The intensity of thescattered light while protein passed through the detector was measuredas a function of angle. This measurement taken together with the proteinconcentration determined using the RI detector allowed calculation ofthe molar mass using appropriate equations (integral part of theanalysis software Astra v.5.3.4.14). The solution state as percentagemonomer is shown in Table 15.

TABLE 15 Percentage monomer DOM23h-439-21 100% DOM23h-439-25 100%DOM23h-439-30 100% DOM23h-439-32 100% DOM23h-439-33 98.2%  DOM23h-439-3497.7%  DOM23h-855-42 83.8%  DOM23h-855-43  83% DOM23h-855-44 64.7% DOM23h-271-123 100% DOM23h-271-124 97.4% 

Example 11 TGFβ-RII Inhibition by Affinity Matured dAbs in the SBE-BlaHEK 293T Cell Sensor Assay

The assay was carried out exactly as outlined in example 4, assay h2.

The assay was performed multiple times to obtain an average and a rangeof values which are summarised in Table 16. The arithmetic mean IC50 wascalculated using log IC50s, and the range was calculated by adding andsubtracting the log standard deviation from the mean IC50, and thentransforming back to IC50. The assay QC parameters were met; the robustZ factors were greater than 0.4 and the TGF-β EC80 was within 6 fold ofthe concentration added to the assay. The results are shown in Table 16.

TABLE 16 Cell Functional assay data for human specific clones plus VHDummy dAb. IC50 nM IC50 range Mean (+/− log SD) n DOM23h-271-123 18.3 9.8-34.1 11 DOM23h-271-129 22.7 16.6-31.1 4 DOM23h-271-130 37.015.1-90.7 4 DOM23h-271-131 6.3 4.5-8.9 4 DOM23h-271-132 21.0 16.1-27.5 4DOM23h-271-133 2.4 1.5-3.8 4 DOM23h-439-25 4.0  1.1-14.7 17DOM23h-439-35 0.5 0.4-0.7 3 DOM23h-439-37 0.7 0.2-2.2 4 DOM23h-439-40 1410.8-18.8 6 DOM23h-439-41 1.7 1.3-2.3 4 DOM23h-439-42 0.7 0.2-2.7 8DOM23h-439-43 1.0 0.7-1.4 4 DOM23h-439-44 1.3 0.5-3.3 6 VHDummy2 >2511925119 13Sequence Concordance Table

SEQ ID NO DOM number Description 1 DOM23h-802 amino acid sequence -naive clone 2 DOM23h-803 amino acid sequence - naive clone 3 DOM23h-813amino acid sequence - naive clone 4 DOM23h-815 amino acid sequence -naive clone 5 DOM23h-828 amino acid sequence - naive clone 6 DOM23h-830amino acid sequence - naive clone 7 DOM23h-831 amino acid sequence -naive clone 8 DOM23h-840 amino acid sequence - naive clone 9 DOM23h-842amino acid sequence - naive clone 10 DOM23h-843 amino acid sequence -naive clone 11 DOM23h-850 amino acid sequence - naive clone 12DOM23h-854 amino acid sequence - naive clone 13 DOM23h-855 amino acidsequence - naive clone 14 DOM23h-865 amino acid sequence - naive clone15 DOM23h-866 amino acid sequence - naive clone 16 DOM23h-874 amino acidsequence - naive clone 17 DOM23h-883 amino acid sequence - naive clone18 DOM23h-903 amino acid sequence - naive clone 19 DOM23m-4 amino acidsequence - naive clone 20 DOM23m-29 amino acid sequence - naive clone 21DOM23m-32 amino acid sequence - naive clone 22 DOM23m-62 amino acidsequence - naive clone 23 DOM23m-71 amino acid sequence - naive clone 24DOM23m-72 amino acid sequence - naive clone 25 DOM23m-81 amino acidsequence - naive clone 26 DOM23m-99 amino acid sequence - naive clone 27DOM23m-101 amino acid sequence - naive clone 28 DOM23m-352 amino acidsequence - naive clone 29 DOM23h-271-21 amino acid sequence - affinitymatured 30 DOM23h-271-22 amino acid sequence - affinity matured 31DOM23h-271-27 amino acid sequence - affinity matured 32 DOM23h-271-101amino acid sequence - affinity matured 33 DOM23h-271-102 amino acidsequence - affinity matured 34 DOM23h-271-105 amino acid sequence -affinity matured 35 DOM23h-271-106 amino acid sequence - affinitymatured 36 DOM23h-271-114 amino acid sequence - affinity matured 37DOM23h-271-39 amino acid sequence - affinity matured plus D61R K64Dmutation 38 DOM23h-271-40 amino acid sequence - affinity matured plusD61R K64F mutation 39 DOM23h-802 nucleic acid sequence - naive clone 40DOM23h-803 nucleic acid sequence - naive clone 41 DOM23h-813 nucleicacid sequence - naive clone 42 DOM23h-815 nucleic acid sequence - naiveclone 43 DOM23h-828 nucleic acid sequence - naive clone 44 DOM23h-830nucleic acid sequence - naive clone 45 DOM23h-831 nucleic acidsequence - naive clone 46 DOM23h-840 nucleic acid sequence - naive clone47 DOM23h-842 nucleic acid sequence - naive clone 48 DOM23h-843 nucleicacid sequence - naive clone 49 DOM23h-850 nucleic acid sequence - naiveclone 50 DOM23h-854 nucleic acid sequence - naive clone 51 DOM23h-855nucleic acid sequence - naive clone 52 DOM23h-865 nucleic acidsequence - naive clone 53 DOM23h-866 nucleic acid sequence - naive clone54 DOM23h-874 nucleic acid sequence - naive clone 55 DOM23h-883 nucleicacid sequence - naive clone 56 DOM23h-903 nucleic acid sequence - naiveclone 57 DOM23m-4 nucleic acid sequence - naive clone 58 DOM23m-29nucleic acid sequence - naive clone 59 DOM23m-32 nucleic acid sequence -naive clone 60 DOM23m-62 nucleic acid sequence - naive clone 61DOM23m-71 nucleic acid sequence - naive clone 62 DOM23m-72 nucleic acidsequence - naive clone 63 DOM23m-81 nucleic acid sequence - naive clone64 DOM23m-99 nucleic acid sequence - naive clone 65 DOM23m-101 nucleicacid sequence - naive clone 66 DOM23m-352 nucleic acid sequence - naiveclone 67 DOM23h-271-21 nucleic acid sequence - affinity matured 68DOM23h-271-22 nucleic acid sequence - affinity matured 69 DOM23h-271-27nucleic acid sequence - affinity matured 70 DOM23h-271-101 nucleic acidsequence - affinity matured 71 DOM23h-271-102 nucleic acid sequence -affinity matured 72 DOM23h-271-105 nucleic acid sequence - affinitymatured 73 DOM23h-271-106 nucleic acid sequence - affinity matured 74DOM23h-271-114 nucleic acid sequence - affinity matured 75 DOM23h-271-39nucleic acid sequence - affinity matured plus D61R K64D mutation 76DOM23h-271-40 nucleic acid sequence - affinity matured plus D61R K64Fmutation 77 DOM23h-802 CDR1 113 . . . CDR2 149 . . . CDR3 78 DOM23h-803CDR1 114 . . . CDR2 150 . . . CDR3 79 DOM23h-813 CDR1 115 . . . CDR2 151. . . CDR3 80 DOM23h-815 CDR1 116 . . . CDR2 152 . . . CDR3 81DOM23h-828 CDR1 117 . . . CDR2 153 . . . CDR3 82 DOM23h-830 CDR1 118 . .. CDR2 154 . . . CDR3 83 DOM23h-831 CDR1 119 . . . CDR2 155 . . . CDR384 DOM23h-840 CDR1 120 . . . CDR2 156 . . . CDR3 85 DOM23h-842 CDR1 121. . . CDR2 157 . . . CDR3 86 DOM23h-843 CDR1 122 . . . CDR2 158 . . .CDR3 87 DOM23h-850 CDR1 123 . . . CDR2 159 . . . CDR3 88 DOM23h-854 CDR1124 . . . CDR2 160 . . . CDR3 89 DOM23h-855 CDR1 125 . . . CDR2 161 . .. CDR3 90 DOM23h-865 CDR1 126 . . . CDR2 162 . . . CDR3 91 DOM23h-866CDR1 127 . . . CDR2 163 . . . CDR3 92 DOM23h-874 CDR1 128 . . . CDR2 164. . . CDR3 93 DOM23h-883 CDR1 129 . . . CDR2 165 . . . CDR3 94DOM23h-903 CDR1 130 . . . CDR2 166 . . . CDR3 95 DOM23m-4 CDR1 131 . . .CDR2 167 . . . CDR3 96 DOM23m-29 CDR1 132 . . . CDR2 168 . . . CDR3 97DOM23m-32 CDR1 133 . . . CDR2 169 . . . CDR3 98 DOM23m-62 CDR1 134 . . .CDR2 170 . . . CDR3 99 DOM23m-71 CDR1 135 . . . CDR2 171 . . . CDR3 100DOM23m-72 CDR1 136 . . . CDR2 172 . . . CDR3 101 DOM23m-81 CDR1 137 . .. CDR2 173 . . . CDR3 102 DOM23m-99 CDR1 138 . . . CDR2 174 . . . CDR3103 DOM23m-101 CDR1 139 . . . CDR2 175 . . . CDR3 104 DOM23m-352 CDR1140 . . . CDR2 176 . . . CDR3 105 DOM23h-271-21 CDR1 141 . . . CDR2 177. . . CDR3 106 DOM23h-271-22 CDR1 142 . . . CDR2 178 . . . CDR3 107DOM23h-271-27 CDR1 143 . . . CDR2 179 . . . CDR3 108 DOM23h-271-101 CDR1144 . . . CDR2 180 . . . CDR3 109 DOM23h-271-102 CDR1 145 . . . CDR2 181. . . CDR3 110 DOM23h-271-105 CDR1 146 . . . CDR2 182 . . . CDR3 111DOM23h-271-106 CDR1 147 . . . CDR2 183 . . . CDR3 112 DOM23h-271-114CDR1 148 . . . CDR2 184 . . . CDR3 185 DOM008 primer 186 DOM009 primer187 DOM172 primer 188 DOM173 primer 189 271-7R1deg CDR1 primer 190271-7R2deg CDR2 primer 191 271-7R3deg CDR3 primer 192 PE008 primer 193271-6164 R primer 194 271-6164 deg-F primer 195 AS1309 primer 196271-6164 NR-F primer 197 DOM57 primer 198 DOM6 primer 199 DOM23h-271amino acid sequence - naive clone 200 DOM23h-271 nucleic acid sequence -naive clone 201 DOM23h-271-7 amino acid sequence - naive clone 202DOM23h-271-7 nucleic acid sequence - naive clone 203 DOM23h-855-21nucleic acid sequence - test matured clone 204 DOM23h-855-21 amino acidsequence - test matured clone 205 DOM23h-843-13 nucleic acid sequence -test matured clone 206 DOM23h-843-13 amino acid sequence - test maturedclone 207 DOM23h-439-20 nucleic acid sequence - test matured clone 208DOM23h-439-20 amino acid sequence - test matured clone 209 PEP-26-FPrimer 210 PelB NcoVh Primer 211 PEP011 Primer 212 DOM-271-50 nucleicacid sequence - CDR-directed affinity matured clone 213 DOM-271-50 aminoacid sequence - CDR-directed affinity matured clone 214 DOM-271-50 aminoacid sequence - CDR-directed affinity matured clone (*duplicate of 213above*) 215 PEP044 Primer 216 23h-439-20 CDRH1 Primer 217 23h-439-20CDRH2 Primer 218 23h-439-20 CDRH3 Primer 219 23h-843-13 CDRH1 Primer 22023h-843-13 CDRH2 Primer 221 23h-843-13 CDRH3 Primer 222 23h-855-21 CDRH1Primer 223 23h-855-21 CDRH2 Primer 224 23h-855-21 CDRH3 Primer 225H1-271-43 R Primer 226 H2p1-271-43 F Primer 227 H2p2-271-43 F Primer 228H3p1-271-43 F Primer 229 H3p2-271-43 F Primer 230 H3p3-271-43 F Primer231 PEP011VHStopNotIR Primer 232 Nco1 VH F Primer 233 DOM23h-439-25nucleic acid sequence - CDR-directed affinity matured clone 234DOM23h-439-25 amino acid sequence - CDR-directed affinity matured clone235 DOM23h-271-123 nucleic acid sequence - CDR-directed affinity maturedclone 236 DOM23h-271-123 amino acid sequence - CDR-directed affinitymatured clone 237 DOM23h-439-35 nucleic acid sequence - CDR-directedaffinity matured clone 238 DOM23h-439-35 amino acid sequence -CDR-directed affinity matured clone 239 DOM23h-271-129 nucleic acidsequence - CDR-directed affinity matured clone 240 DOM23h-271-129 aminoacid sequence - CDR-directed affinity matured clone 241 DOM23h-271-123CDR1 242 DOM23h-271-123 CDR2 243 DOM23h-271-123 CDR3 244 DOM23h-271-129CDR1 245 DOM23h-271-129 CDR2 246 DOM23h-271-129 CDR3 247 DOM23h-439-25CDR1 248 DOM23h-439-25 CDR2 249 DOM23h-439-25 CDR3 250 DOM23h-439-35CDR1 251 DOM23h-439-35 CDR2 252 DOM23h-439-35 CDR3 253 439 48I SDM FPrimer 254 439 61N SDM F Primer 255 439 64R SDM F Primer 256 439 61N 64RSDM F Primer 257 271 61N SDM F Primer 258 271 64R SDM F Primer 259 27161N 64R SDM F Primer 260 567 +A rev Primer 261 21-23 Fwd Primer 262DOM23h-439-40 nucleic acid sequence - CDR-directed affinity maturedclone 263 DOM23h-439-40 amino acid sequence - CDR-directed affinitymatured clone 264 DOM23h-439-41 nucleic acid sequence - CDR-directedaffinity matured clone 265 DOM23h-439-41 amino acid sequence -CDR-directed affinity matured clone 266 DOM23h-439-42 nucleic acidsequence - CDR-directed affinity matured clone 267 DOM23h-439-42 aminoacid sequence - CDR-directed affinity matured clone 268 DOM23h-439-43nucleic acid sequence - CDR-directed affinity matured clone 269DOM23h-439-43 amino acid sequence - CDR-directed affinity matured clone270 DOM23h-439-44 nucleic acid sequence - CDR-directed affinity maturedclone 271 DOM23h-439-44 amino acid sequence - CDR-directed affinitymatured clone 272 DOM23h-271-130 nucleic acid sequence - CDR-directedaffinity matured clone 273 DOM23h-271-130 amino acid sequence -CDR-directed affinity matured clone 274 DOM23h-271-131 nucleic acidsequence - CDR-directed affinity matured clone 275 DOM23h-271-131 aminoacid sequence - CDR-directed affinity matured clone 276 DOM23h-271-132nucleic acid sequence - CDR-directed affinity matured clone 277DOM23h-271-132 amino acid sequence - CDR-directed affinity matured clone278 DOM23h-271-133 nucleic acid sequence - CDR-directed affinity maturedclone 279 DOM23h-271-133 amino acid sequence - CDR-directed affinitymatured clone 280 DOM23h-271-134 nucleic acid sequence - CDR-directedaffinity matured clone 281 DOM23h-271-134 amino acid sequence -CDR-directed affinity matured clone 282 DOM23h-271-135 nucleic acidsequence - CDR-directed affinity matured clone 283 DOM23h-271-135 aminoacid sequence - CDR-directed affinity matured clone 284 DOM23h-271-136nucleic acid sequence - CDR-directed affinity matured clone 285DOM23h-271-136 amino acid sequence - CDR-directed affinity matured clone286 DOM23h-271-137 nucleic acid sequence - CDR-directed affinity maturedclone 287 DOM23h-271-137 amino acid sequence - CDR-directed affinitymatured clone 288 DOM23h-439-47 nucleic acid sequence - DOM23h-439-42 +48I 289 DOM23h-439-47 amino acid sequence - DOM23h-439-42 + 48I 290DOM23h-439-48 nucleic acid sequence - DOM23h-439-44 + 48I 291DOM23h-439-48 amino acid sequence - DOM23h-439-44 + 48I

The invention claimed is:
 1. An anti-TGFbetaRII immunoglobulin singlevariable domain comprising the amino acid sequence of SEQ ID NO:
 267. 2.An anti-TGFbetaRII immunoglobulin single variable domain as claimed inclaim 1, wherein the anti-TGFbetaRII immunoglobulin single variabledomain further comprises a C-terminal alanine residue.
 3. Ananti-TGFbetaRII immunoglobulin single variable domain as claimed inclaim 1, wherein said anti-TGFbetaRII immunoglobulin single variabledomain binds to human TGFbetaRII.
 4. An anti-TGFbeta RII immunoglobulinsingle variable domain as claimed in claim 3, wherein saidanti-TGFbetaRII immunoglobulin single variable domain also binds tomouse TGFbetaRII and/or cyno TGFbetaRII.
 5. An immunoglobulin singlevariable domain as claimed in claim 1, wherein said immunoglobulinsingle variable domain neutralises TGFbeta activity.
 6. Animmunoglobulin single variable domain as claimed in claim 1, whereinsaid immunoglobulin single variable domain inhibits binding of TGFbetato TGFbetaRII.
 7. A pharmaceutical composition comprising ananti-TGFbetaRII immunoglobulin single variable domain comprising anamino acid sequence as set forth in SEQ ID NO: 267 for use in treatingtissue fibrosis or for use in wound healing and/or scarring reduction.8. A pharmaceutical composition comprising an anti-TGFbetaRIIimmunoglobulin single variable domain comprising an amino acid sequenceas set forth in SEQ ID NO: 267 for use in treating keloid disease orDupuytren's Contracture.